Background Serratia marcescens becomes an apparent nosocomial pathogen and causes a variety of infections. S. marcescens possess various virulence factors that are regulated by intercellular communication system quorum sensing (QS). Targeting bacterial virulence is a proposed strategy to overcome bacterial resistance. Sitagliptin anti-QS activity has been demonstrated previously and we aimed in this study to investigate the effects of antidiabetic drugs vildagliptin and metformin compared to sitagliptin on S. marcescens pathogenesis. Methods We assessed the effects of tested drugs in subinhibitory concentrations phenotypically on the virulence factors and genotypically on the virulence encoding genes' expressions. The protection of tested drugs on S. marcescens pathogenesis was performed in vivo. Molecular docking study has been conducted to evaluate the interference capabilities of tested drugs to the SmaR QS receptor. Results Vildagliptin reduced the expression of virulence encoding genes but did not show in vitro or in vivo anti-virulence activities. Metformin reduced the expression of virulence encoding genes and inhibited bacterial virulence in vitro but did not show in vivo protection. Sitagliptin significantly inhibited virulence factors in vitro, reduced the expression of virulence factors and protected mice from S. marcescens. Docking study revealed that sitagliptin is more active than metformin and fully binds to SmaR receptor, whereas vildagliptin had single interaction to SmaR. Conclusion The downregulation of virulence genes was not enough to show anti-virulence activities. Hindering of QS receptors may play a crucial role in diminishing bacterial virulence.