<p>Benzalkonium chloride (BAC) is a widely used quaternary ammonium compound whose persistence in wastewater systems raises concerns regarding its biodegradation and the associated microbial adaptive responses. This study examines the effect of carbon substrate composition on BAC biodegradation, microbial community structure, and antimicrobial susceptibility in semicontinuous activated sludge systems exposed to sub-inhibitory BAC concentrations. Reactors were operated using either a proteinaceous substrate (peptone) or a readily biodegradable substrate (acetate). Clear differences in the system's response were observed depending on the carbon source supplied. Peptone-fed systems sustained microbial activity and promoted effective BAC biodegradation, whereas acetate-fed systems exhibited pronounced toxicity, reduced biological activity, and negligible BAC removal. These contrasting behaviors were accompanied by marked shifts in microbial community diversity and composition. In parallel, exposure to BAC was associated to changes in antimicrobial susceptibility patterns, particularly affecting β-lactam antibiotics, that could be attributed to a substrate-dependent selective pressure. These results shows that carbon substrate composition may influence BAC biodegradation capacity and microbial adaptation in activated sludge systems. The findings contribute to a better understanding of the factors potentially controlling the biodegradation of quaternary ammonium compounds and highlight the important role of carbon availability in shaping microbial responses under BAC-associated selective stress.</p>