Dr. James Marcus, the founder of the Umbrella Corporation, had a daughter, Alicia, who was dying of progeria. Desperate to save her, Marcus uses the T-virus on her and others with the disease. After another child treated with the virus died and then reanimated as a rabid zombie, Marcus tried to have the program shut down. His partner, Dr. Alexander Isaacs, had Marcus killed by Albert Wesker, then adopted Alicia and took over the Corporation.

安布雷拉公司[傘公司]的創始人，詹姆斯·馬庫斯博士有個女兒艾莉西亞正在早衰死亡。馬庫斯幾乎絕望，把T病毒用于給女兒和患有該病的別人治療。在對另一個孩子施用該病毒治療后，患者死掉又活過來立即成了僵尸，馬庫斯為此企圖關閉該流程。他的商業伙伴亞歷山大·艾薩克斯命令阿爾伯特·威斯克處死了馬斯克，自己收養了艾莉西亞并接管了該公司。

After the events in Washington D.C., Alice awakens after being betrayed by Wesker. The Red Queen appears and tells Alice she has 48 hours to infiltrate the Hive, over 400 miles west on I-64 in Raccoon City. The Umbrella Corporation has developed an airborne anti-virus, which can kill every organism infected by the T-virus, but is waiting for the last few remaining humans to be wiped out.

哥倫比亞特區華盛頓事件后，愛麗絲經歷威斯克的背叛后蘇醒過來。紅后出現并告知愛麗絲，她必須在48小時內潛入浣熊市蜂巢，蜂巢位于向西400英里之外的浣熊市I-64區。安布雷拉公司已經開發出了能空氣傳播的抗病毒，會殺死所有感染了T病毒的生物，目前正等著最后遺留的少數人類被消滅。

Enroute to Raccoon City, Alice is captured by Isaacs, learning the "Isaacs" she previously killed was a clone. She escapes his convoy and reaches Raccoon City, where she is ambushed by a group of survivors: Doc, Abigail, Christian, Cobalt, Razor, and Claire Redfield, who survived the attack on Arcadia. Isaacs' convoy approaches, trailed by a horde of zombies. Alice and the group defeat the convoy, retrieve Isaacs' few human captives and burn the horde, though Cobalt is killed. Alice and crew enter the Hive entrance, at the bottom of the crater from the nuclear bomb previously dropped on the city. Wesker, in control of the Hive, releases mutated guard dogs, killing Christian and a freed captive.

在去往浣熊市的路上，愛麗絲被艾薩克斯俘虜，認識到她原來殺死的那個艾薩克斯其實是個克隆人。愛麗絲逃脫他的護衛隊到達浣熊市后被一群幸存者伏擊，這些幸存者有道克、艾比蓋爾、克里斯托、克波特、瑞澤、克萊爾·雷德菲爾德，他們是阿卡迪亞遭到攻擊后的幸存者。艾薩克斯護衛隊趕往這里，尾隨了一大群僵尸。愛麗絲隊伍擊敗了護衛隊，收容了艾薩克斯的幾個人類俘虜，焚毀了僵尸群，但克波特在戰斗中喪生。愛麗絲隊伍進入蜂巢入口，入口在之前浣熊市丟核彈炸的大坑底部。威斯克控制著蜂巢，釋放了變異的警衛狗，殺死了克里斯托和一個自由了的俘虜。

The Red Queen appears to Alice and explains that her program is in conflict, as she can never hurt an Umbrella employee but also must value human life. She plays a video of Isaacs explaining to Umbrella's executives a plan to release the T-virus, cleansing the world of humanity; many of the rich and powerful, including the company executives, are stored in cryogenic capsules in the Hive, with the intention of rebuilding the world following the resulting apocalypse. The Red Queen warns Alice that someone in her group is helping Umbrella.

紅后出現在愛麗絲面前，解釋她的程序有沖突，因為她不能傷害安布雷拉雇員而且還要衡量人類生命。紅后播放了一個影碟，艾薩克斯正給安布雷拉公司管理層解釋他的計劃，釋放T病毒清理人類世界，很多富人和當權者包括公司管理者，用低溫莢膜封藏在蜂巢里，毀滅世界后再重建。紅后警告愛麗絲，她的隊伍里有人正在幫助愛布雷拉公司。

The group encounters several traps, killing Abigail and Razor. Alice and Doc plant bombs throughout the Hive, from the leftover equipment of the original team to investigate the Hive. Alice confronts the real, tech-upgraded, Isaacs. Doc is revealed to be Umbrella's spy, and Claire is captured by Wesker. A cryogenic capsule opens, releasing Alicia Marcus, Umbrella's co-owner, and Marcus' daughter. Isaacs reveals to Alice that she is actually a clone of Alicia, before going on to explain his plan to eliminate the pair and assume control of Umbrella. Alicia fires Wesker, allowing the Red Queen to crush his legs with a security door. Doc tries to shoot Alice, but his gun is empty — as Alice had earlier deduced his treachery — and Claire executes him. After giving Wesker a deadman's switch to the primed bombs, Alice and Claire pursue Isaacs while Alicia uploads a copy of her childhood memories.
這群人遭遇了幾個陷阱，艾比蓋爾和瑞澤喪生。愛麗絲和道克在整個蜂巢部署了炸彈，從原隊伍的遺留設備處一直到勘察蜂巢沿路[譯注：原文有錯誤]。愛麗絲面對真實有技術裝備的艾薩克斯。道克被暴出是安布雷拉安插[在幸存者中]的間諜，克萊爾也被威斯克抓住。一個低溫莢膜打開，艾麗西亞·馬庫斯 出現，她是公司的合伙持有人，詹姆斯·馬庫斯的女兒。艾薩克斯首先揭示愛麗絲是艾莉西亞的克隆人，然后解釋他計劃除掉合伙人艾莉西亞完全掌控愛布雷拉。艾莉西亞煽動威斯克，讓紅后關閉安全門夾斷他的腿。道克向愛麗絲開槍，但沒有子彈--愛麗絲之前推斷出他是叛徒--克萊爾擊斃了道克。塞給威斯克一個死人開關[譯注：不按著蜂巢就炸]后，愛麗絲和克萊爾追殺艾薩克斯，此刻艾莉西亞上傳她童年記憶的拷貝。

Isaacs, Alice and Claire fight; Isaacs overpowers Claire and attacks Alice in the Laser Corridor, eventually defeating her, but she uses that moment to activate a grenade in his pocket. She escapes to the surface with the anti-virus, but Isaacs reboots and catches her before she can release it. Before he can kill her, the Isaacs clone (from the convoy) arrives and kills him, believing itself to be the original Isaacs, but the clone is then devoured by the undead. Alice releases the anti-virus, killing all of the undead around her before she passes out. Wesker simultaneously drops the deadman's switch, destroying himself, Alicia, the Hive, and the hibernating Umbrella elite.

艾薩克斯對抗愛麗絲和克萊爾，艾薩克斯戰勝克萊爾，并在激光走廊里攻擊愛麗絲最終獲勝，但愛麗絲利用該時機觸發了他口袋里的手雷。愛麗絲帶著抗病毒逃到地面，但艾薩克斯復活并在愛麗絲投放抗病毒前抓住了她。在艾薩克斯殺死愛麗絲前，艾薩克斯的克隆人(來自護衛隊)趕到，殺死了艾莎克斯，覺得自己才是真正的艾薩克斯，但克隆人又被僵尸吞沒。愛麗絲釋放了抗病毒，她倒下前殺死了她周圍所有的僵尸。此刻，威斯克丟下了死人開關，毀掉了自己，艾莉西亞，蜂巢，休眠中的安布雷拉精英們。

Claire wakes Alice, who survived because the anti-virus killed only the T-virus within her body, not the healthy cells. The Red Queen uploads Alicia's childhood memories into Alice, granting her a childhood. Alice travels into Manhattan, saying that the anti-virus, carried only by the winds, will take years to reach all corners of the globe, and until it does, her mission is not finished.

克萊爾喚醒了愛麗絲，愛麗絲活了下來，因為抗病毒只殺死她體內的T病毒，而非健康細胞。紅后給愛麗絲加載了艾莉西亞所有的童年記憶，給她賦予了童年。愛麗絲去曼哈頓旅行，[故事]聲稱抗病毒被風吹到世界各角落需要多年，在此結果前，愛麗絲的任務還沒結束。