Background: Withania somnifera (WS) is proposed as one of the alternatives instead of the antibiotic. This study is aimed to evaluate the inhibitory potency of enzymatic extract of the fruits of the WS. Methods: As an invitro experimental study, the growth rate of Shigella dysenteriae, Salmonella typhimurium, and Escherichia coli inoculated in different concentrations (25%, 12.5%, 6.25% and 3.125%) of the extract were assessed. A microtitre plate method was conducted. ANOVA was applied to identify statistical differences with p-value <0.05). Compared to the microorganism control, effective concentration of the extract inhibiting the growth of E. coli was 12.5%, and 6.25%, while it was 12.5%, and 6.25% for Sh. dysenteriae (p<0.05). A dose-dependent response of E. coli was observed. The antibacterial activity of the extract tested was found mainly against E. coli and Sh. dysenteriae. The most resistant microorganism compared to E. coli and Sh. dysenteriae was S. typhimurium (p<0.05). 25% of the concentration of the extract showed the different inhibitory effect among three tested bacteria (p<0.05). Conclusions: The extract was labeled as an antibacterial agent against the representative of three foodborne bacteria, Invitro. The common effective concentrations of the extract (12.5, and 6.25%) is recommended for further research, as food additive, to remedy digestive ailments related to E. coli, S. typhimurium and Sh. dysenteriae