Pseudomonas aeruginosa, a formidable opportunistic pathogen, is notorious for its ability to form biofilms and produce virulence factors that favor chronic infections, especially in cystic fibrosis patients. The misuse of disinfectants, combined with environmental leakage and biodegradation, has led to widespread exposure of microorganisms to sub-lethal concentrations of disinfectants, particularly quaternary ammonium compounds (QACs). This study investigates the interaction between QACs, specifically ethylbenzalkyl dimethyl ammonium chloride (EBAC), and the quorum sensing (QS) mechanisms governing P. aeruginosa behavior. The results demonstrate that exposure to sub-minimum inhibitory concentrations (sub-MICs) of EBAC not only enhances the biofilm-forming capability of P. aeruginosa isolates but also modulates the expression of crucial QS-regulated genes. Notably, the bacteria exhibit increased production of biofilm-associated virulence factors such as pyocyanin and elastase, and altered antibiotic susceptibility profiles, indicating a shift towards persistent infection phenotypes. These findings reveal that QAC exposure can significantly increase resistance to antibiotics and external stressors like hydrogen peroxide. These results emphasize the need to reassess the efficacy of QACs in clinical disinfection settings, particularly against P. aeruginosa infections, and highlight the potential for unintended consequences of their use regarding bacterial behavior and virulence. This study provides novel insights into the role of QACs in modulating QS-mediated virulence and antibiotic resistance, offering a new perspective on the risks associated with sub-lethal disinfectant exposure.