Antimicrobial biocides – used as disinfectants, antiseptics and preservatives – are essential for infection control and prevention of contamination in many different settings. Unlike antibiotics, biocides are not subject to stewardship efforts to monitor and preserve their efficacy. Despite their importance and widespread use, we have very limited knowledge of the precise mechanisms by which these agents kill microbes, or the long-term consequences of repeated microbial exposure. We conducted the first broad genome-scale screen for biocide survival, using the frequently disinfectant-resistant ESKAPE pathogen Acinetobacter baumannii as the target organism. The majority of biocides tested had defined intracellular targets, including genes involved in various processes not previously linked to biocide survival. Nine of ten biocides dissipated membrane potential when present at subinhibitory concentrations, and this was linked to antagonism of several classes of antibiotics. Further investigations of one biocide-antibiotic class pair (benzalkonium chloride and aminoglycosides) showed that this antagonism protects bacteria from killing and dramatically increases the emergence of mutants with reduced susceptibility. Overall our results suggest that indiscriminate biocide use could be a hidden contributor to the antibiotic resistance crisis, and that exposure of bacteria to subinhibitory levels of biocides (for example those found as preservatives in pharmaceutical products) should be avoided.