A total of 34 bacterial strains with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity were isolated from 69 soil and water samples collected from four areas of Thailand. One strain, WAPB4 identified as Bacillus pumilus, showed remarkable antibacterial activity against MRSA, vancomycin-resistant Enterococcus faecalis (VRE), and several Gram-positive test bacteria. Bacteriocin produced by WAPB4 was designated as pumilicin 4. It was heat stable up to 121 degrees C, 15 min and active within the pH range of 3-9. Its activity disappeared when treated with pronase E, chymotrypsin, and trypsin, demonstrating its proteinaceous nature. At high dosage (80 AU mL(-1)), the effect of pumilicin 4 was bactericidal to both MRSA and VRE. Bacteriostasis was observed for a low dose of bacteriocin (20 AU mL(-1)). Purification of pumilicin 4 was performed by a three-step procedure, i.e., solvent extraction, solid phase extraction, and reversed-phase chromatography. The molecular mass of purified pumilicin 4 as determined by mass spectrometry was 1994.62 Dalton. This present study is the first report of a novel bacteriocin, pumilicin 4, produced by B. pumilus that has potential for use as an alternative antibacterial agent for the treatment of infection with MRSA and VRE.