In Gram-negative bacteria, inhibition of the disulfide bond (DSB) oxidative protein folding machinery -a major facilitator of bacterial virulence- is considered an attractive antivirulence strategy. We have previously developed small molecule inhibitors of the prototypic DsbA enzyme from Escherichia coli K-12 (EcDsbA) and showed that they can block DsbA homologues found in pathogens. EcDsbA inhibitors were active against uropathogenic E. coli (UPEC) and Salmonella enterica, both of which contain more than one copy of DsbA. Our inhibitors attenuated UPEC and S. Typhimurium virulence without affecting their growth in rich medium. Here we test the evolutionary robustness of DsbA inhibitors as antivirulence antimicrobials by looking for resistance development in S. Typhimurium in physiologically relevant in vitro conditions. We demonstrated that DsbA inhibitors had a fitness cost on S. Typhimurium, cultured in physiologically relevant conditions, a phenotype that was DsbA-specific, as a dsbA null mutant cultured under the same conditions had a similar growth defect. Despite the fitness cost of DsbA inhibitors on S. Typhimurium growth, no inhibitor resistance was detected following treatment at sub-IC50 concentrations. No mutations were identified in the target gene and all inhibitor-treated S. Typhimurium culture displayed DsbA-mediated virulence phenotypes similar to the untreated pathogen. Importantly, these phenotypes remained sensitive to inhibition by DsbA inhibitors to the same extent as bacteria not previously exposed to inhibitor. Our work provides in vitro evidence that DsbA inhibitors do not select for resistant mutants in physiologically relevant conditions, suggesting that they could offer evolutionary robust new antimicrobials to be used instead or in combination with antibiotics.