The aim of this study was to develop in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella spp. in the presence of Phe-Arg β-naphthylamide (PAβN) and to observe which AZM resistance mechanisms other than efflux pumps were inhibited by PAβN emerge. The frequency of mutation ranged between <6.32 × 10^−10 and 5.22 × 10^−7 for E. coli and between <5.32 × 10^−10 and 1.69 × 10^−7 for Shigella spp. The E. coli mutants showed an increase in the AZM minimum inhibitory concentration (MIC) up to 128-fold, whilst the Shigella spp. mutants presented increases in MIC levels of up to 8-fold. In one mutant, the insertion of nucleotides encoding the amino acid sequence IMPRAS was found in the rplV gene. Increases in OmpW expression were observed in all E. coli mutants compared with their respective parental isolates. The combination of antibiotics and efflux pump inhibitors appears to be a good option to reduce the frequency of mutation in clinical isolates.