AMR genes, including multidrug efflux pumps, evolved long before the ubiquitous use of antimicrobials in medicine and infection control. Understanding their ancestral physiological roles through defining their natural substrates could inform the development of strategies to subvert their activity. Polyamines are an ancient, widespread class of amino acid-derived metabolites with some structural similarity to widely used disinfectants. In this study, we investigated the effects of polyamines on the priority bacterial pathogen Acinetobacter baumannii, which has a particularly striking ability to survive disinfectant treatment due to its arsenal of multidrug efflux pumps. Polyamine shock induced expression of numerous genes in A. baumannii including amvA, which encodes a disinfectant efflux pump of the MFS family. Loss of amvA dramatically reduced tolerance of A. baumannii to long-chain polyamines, while overexpression in E. coli promoted polyamine tolerance. A role for AmvA in polyamine tolerance was shown in A. baumannii strains from both of the dominant global lineages of this pathogen. We also demonstrate that long-chain polyamines induce expression of amvA through binding to its cognate repressor, AmvR. Taken together, our results indicate that polyamines are very likely to be the natural substrates of the AmvA efflux pump. Our results suggest that the disinfectant resistance capability that allows A. baumannii to survive in hospitals may have evolutionary origins in the transport of polyamine metabolites.