In this investigation, the Sepestan fruit mucilage (SFM) was extracted by water to seed ratio 1: 6, extraction temperature 58 ᵒ C, and pH 4. After sterilization of extracted mucilage, its antibacterial activity was assessed on pathogenic bacteria including Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, Listeria innocua, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis“ in vitro” . Antimicrobial potential of mucilage, at the different SFM levels: 37. 5, 75, 150 and 300 mg/ml was evaluated using disk diffusion agar and well diffusion agar methods. Furthermore, the interaction of SFMwith gentamicin and chloramphenicol antibiotics was assessed. Results shown that by increasing the SFMconcentrations, the inhibition zone diameter (IZD) around the discs were noticeably increased. In the disk diffusion agar method, the highest IZDwas observed at the concentration of 300 mg/ml SFM for Staphylococcus epidermidis with 11. 10 mm. The minimum inhibitory concentration (MIC) for Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, Listeria innocua, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis was 64, 64, 16, 32, 128, 64, 256 and 64 mg/ml, respectively. Results shown that the minimum bactericidal concentration (MBC) of SFM for the all tested bacterial strains was greater than their MIC.