Abstract Objectives To systematically analyse the interplay between the expression of Hfq-associated small non-coding RNAs (sRNAs) and antibiotic susceptibility in Gram-negative bacteria. Methods To identify the roles of sRNAs in the antibiotic susceptibility of Escherichia coli and Salmonella species, susceptibility tests, growth analyses and viability assays were performed using E. coli Hfq-associated sRNAs from overexpression libraries. Prediction, susceptibility testing of gene knockouts and expression analysis of target genes under conditions of sRNA overexpression or knockout were performed to identify candidate targets for modulating antibiotic susceptibility. Results The susceptibilities of E. coli strains overexpressing each of the 26 known Hfq-dependent sRNAs to major classes of antibiotics were determined. Induced expression of 17 sRNAs modulated the susceptibility of E. coli to antibiotics. Among them, four sRNA knockout strains partially or completely reversed susceptibility phenotypes of sRNA overexpression. The phenotype of OxyS, RseX or MicF was not entirely dependent on the presence of Hfq protein, in contrast to the dependency of previously characterized roles. The function of eight of nine sRNAs was found to be conserved in the response to antibiotics in Salmonella. Some MicF- or RyeB-mediated cellular target genes and pathways that may be important for the regulation of antibiotic susceptibility were identified. Finally, the overexpression of RyeB potentiated the efficacy of levofloxacin against MDR strains. Conclusions Our data indicate that Hfq-associated sRNAs potentially enable bacteria to adapt to antibiotic challenges via multifaceted approaches. Therefore, sRNA-based applications will form a new antibiotic arsenal for combating the rise in antibiotic resistance.