Third-generation EGFR tyrosine kinase inhibitors (TKIs), such as osimertinib, have significantly improved outcomes for patients with EGFR-mutant non-small cell lung cancer (NSCLC). However, nearly all patients eventually develop acquired resistance after an initial response, highlighting the urgent need for novel therapeutic strategies to overcome this challenge. Dequalinium Chloride (DQC) is a clinically used cationic amphiphilic antimicrobial agent with low toxicity that selectively accumulates in mitochondria and induces mitochondrial dysfunction. Leveraging these properties, we investigated the therapeutic potential of combining DQC with osimertinib to overcome resistance in EGFR-mutant NSCLC models. The DQC-osimertinib combination synergistically reduced cell viability, suppressed colony formation, and induced Bim-mediated apoptosis in resistant cell lines. In xenograft models, the combination markedly inhibited tumor growth without increasing systemic toxicity. Mechanistically, in osimertinib-sensitive cells, osimertinib alone increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potential, whereas these effects were absent in resistant cells, indicating mitochondrial adaptation during acquired resistance. Co-treatment with DQC and osimertinib induced profound mitochondrial dysfunction, characterized by elevated ROS accumulation, loss of mitochondrial membrane potential, and increased gamma-H2AX foci formation indicating ROS-dependent DNA damage. The ROS scavenger N-acetylcysteine (NAC) attenuated mitochondrial dysfunction and apoptosis, confirming a ROS-mediated mechanism. Moreover, DQC combined with osimertinib synergistically reduced the survival of EGFR-mutant NSCLC cell lines with primary resistance to osimertinib. In osimertinib-sensitive models, the combination also suppressed proliferation and delayed the onset of acquired resistance. Collectively, these findings demonstrate that targeting mitochondrial homeostasis with DQC enhances osimertinib efficacy and delays resistance, supporting a mitochondria-targeted therapeutic approach for EGFR-mutant NSCLC.