Biocides, such as antiseptics and disinfectants, are used ubiquitously for hygiene in households and for life-saving infection control in hospitals. An increasing concern is the widespread use of biocides may contribute to the emergence and spread of multidrug-resistant bacteria. For the first time, we performed transposon directed insertion site sequencing (TraDIS) to identify genes or key cellular pathways of the multidrug resistant nosocomial pathogen Acinetobacter baumannii affecting host fitness during exposure to a panel of ten structurally-diverse and clinically-relevant biocides: silver nitrate, benzalkonium, cetyltrimethylammonium bromide (CTAB), chlorhexidine, triclosan, chloroxylenol, polyvidone iodine, bleach, glutaraldehyde and ethanol. We identified a number of electron transport genes as contributing to tolerance of most biocide treatments, and showed that these biocides caused a drop in cell membrane potential even at sub-inhibitory concentrations; based on these findings, we propose that collapse of membrane potential is a common direct or downstream biocidal mode of action. In addition, the core antibiotic efflux systems in A. baumannii, i.e. AdeABC and AdeIJK, confer resistance to half of the biocides, suggesting these compounds have the potential for co-selection of resistance to the multiple antibiotics that are also transported by these pumps.