Researchers have focused on natural medicines to treat infectious diseases due to antibiotic resistance. Due to the medicinal importance of Datura stramonium and Prosopis farcta, the present study has investigated their antimicrobial effects against some gram-positive and negative bacteria by disc diffusion, micro broth dilution, and agar well diffusion methods and also their possible toxicity using MTT assay against HT29 cells. Additionally, the inhibitory effect of the extracts on the formation of bacterial biofilms was investigated. The hydroalcoholic extract of D. stramonium and P. fracta had antibacterial effects on the bacteria in both micro broth dilution and agar well diffusion methods. D.stramonium and P.fracta had the greatest effects on S.aureus and P.aeruginosa. Their weakest effects were seen towards K. pneumonia and A. baumannii. Anti-biofilm property was observed only for D. stramonium extract (50 mg/ml concentration). MTT assay showed that exposure of HT29 cells to concentrations of 25, 12.5, 6.25, and 3.1 mg/ml of D.stramonium and P.fracta extracts did not cause obvious toxicity. P.farcta and D.stramonium inhibited the growth of bacteria as well as biofilm formation in these strains, and they were not toxic to human cells. Trials on patients using these extracts are recommended, however