A strain named Bacillus velezensis SW5 with multiple enzyme activities of proteinase and carbohydrase, was isolated from the spontaneous fermented marine fish sauce. To investigate the antimicrobial properties of Bacillus velezensis SW5, the growth characteristics and physiological-biochemical characteristics of strain SW5 were investigated. The antimicrobial ability of supernatant against various common pathogenic bacteria in food was verified, and its antimicrobial components were further determined by liquid chromatography-mass spectrometry method. The results showed that 35℃ and pH 5 were the optimum conditions for the growth of strain SW5. The strain SW5 was sensitive to β-lactam antibiotics, including imipenem and ceftriaxone. The antibacterial activity of supernatant of strain SW5 culture was well observed, and it became stronger after being concentrated. And the results of TOF-MS detection indicated that antibacterial substance of supernatant contained surfactin, corchoroside A, aminoglycosides antibiotics gentamicin and isepamicin, streptomycin antibiotics dalfopristin as well. The results aimed to provide a certain theoretical basis for the further application of strain SW5.