Background and Aim : Probiotics may be a good alternative to antibiotics in fighting pathogenic bacteria. The current study aimed to evaluate the antibacterial activity of Lactobacillus rhamnosus cell-free supernatant against Staphylococcus aureus and Escherichia coli. Methods : L. rhamnosus ATCC 7469 was cultured in the MRS broth medium at 37˚C. The medium was transferred to the tube after 48 h and centrifuged at 8000 rpm for 20 minutes. The supernatant was passed through a filter with 0.2 μm pores under sterile conditions. The antibacterial activity of the filtered supernatant was determined against S. aureus ATCC 25923 and E. coli ATCC 8739 by minimum inhibitory concentration (MIC) method. Results : The minimum inhibitory concentration of L. rhamnosus cell-free supernatant against S. aureus and E. coli was 25 and 12.5 microliter/ml, respectively. Conclusion : The cell-free supernatant of L. rhamnosus had a good antibacterial effect on S. aureus and E. coli.