Background: Recently re-emerging infections occur due to antibiotic-resistant bacteria. The use of bacteriocins as a substitute for currently used antibiotics is promising. The isolated nitrogen-fixing Bacillus bacteria might be a potential candidate strain as an alternative source of peptide antibiotics in the future. Methods: A total of 15 nitrogen-fixing Bacillus strains with antibacterial activity were isolated from soil samples. Bacteriocin-producing strains against the indicator strains were co-cultured and isolated. The antibacterial activity of bacteriocin was detected by spot-on-the lawn method with different bacteria. Effects of proteolytic enzymes, pH, and heat treatment on bacteriocin activity were also determined. Findings: All isolated spore forming Bacillus strains had an inhibitory effect on Listeria monocytogenes and Staphylococcus aureus. Some of them had an inhibitory effect on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). None of them had an inhibitory effect on Gram-negative bacteria. The antimicrobial compounds produced by these novel strains were inactivated by the proteolytic enzymes which demonstrated their proteinaceous nature. These bacteriocins were also active in a wide range of pH and temperature. Conclusion: In the present study, new bacteriocin-producing nitrogen-fixing Bacillus strains with anti-MRSA and anti-VRE activity were isolated from the environment and their biochemical properties were determined. Keywords: Nitrogen-fixing Bacillus strains, Bacteriocin, Methicillin-resistant Staphylococcus aureus, Vancomycin-resistant Enterococcus faecalis