BACKGROUND: Combinatorial systemic chemotherapy is a powerful treatment paradigm against cancer, but it is fraught with problems due to the emergence of chemoresistance and additive systemic toxicity. In addition, coadministration of individual drugs suffers from uncontrollable pharmacokinetics and biodistribution, resulting in suboptimal combination synergy. METHODS: Toward the goal of addressing these unmet medical issues, we describe a unique strategy to integrate multiple structurally disparate drugs into a self-assembling nanococktail platform. Conjugation of a polyunsaturated fatty acid (e.g., linoleic acid) with two chemotherapies generated prodrug entities that were miscible with tunable drug ratios for aqueous self-assembly. In vitro and in vivo assays were performed to investigate the mechanism of combinatorial nanococktails in mitigating chemoresistance and the efficacy of nanotherapy. RESULTS: The coassembled nanoparticle cocktails were feasibly fabricated and further refined with an amphiphilic matrix to form a systemically injectable and PEGylated nanomedicine with minimal excipients. The drug ratio incorporated into the nanococktails was optimized and carefully examined in lung cancer cells to maximize therapeutic synergy. Mechanistically, subjugated resistance by nanococktail therapy was achieved through the altered cellular uptake pathway and compromised DNA repair via the ATM/Chk2/p53 cascade. In mice harboring cisplatin-resistant lung tumor xenografts, administration of the nanococktail outperformed free drug combinations in terms of antitumor efficacy and drug tolerability. CONCLUSION: Overall, our study provides a facile and cost-effective approach for the generation of cytotoxic nanoparticles to synergistically treat chemoresistant cancers.