Synthetic amphiphiles have emerged as potent bactericidal scaffolds owing to their high propensity to interact with bacterial cells and a membrane-directed mode of action, which is likely to overcome resistance development in pathogenic bacteria. In this study, we highlighted a membrane-acting quinolinium-based cationic amphiphile (compound 1) as an adjuvant for antibiotic-mediated eradication of pathogenic bacteria and demonstrated the generation of an amphiphile-loaded nanocarrier for potential antibacterial therapy. Treatment of Gram-negative pathogenic bacteria E. coli MTCC 433 and P. aeruginosa MTCC 2488 with 1 resulted in significant augmentation of the activity of erythromycin and a decrease in the minimum inhibitory concentration of the antibiotic. Interestingly, 1 promoted large-scale eradication of P. aeruginosa MTCC 2488 biofilm and could also enhance the anti-biofilm activity of tobramycin in combination. For potential therapeutic applications, a 1-loaded bovine serum albumin-based nanocarrier was developed, which exhibited sustained release of 1 both in physiological and acidic pH and the released amphiphile displayed antibacterial as well as anti-biofilm activities. Interestingly, the nanocarrier also displayed the signature membrane-directed activity of 1 against tested pathogenic bacteria. The high bactericidal and anti-biofilm activities in conjunction with a lack of cytotoxic effect on HT-29 human cell lines enhance the merit of the amphiphile-loaded nanocarrier as a potentially therapeutic antibacterial against clinically relevant drug-resistant pathogenic bacteria.