A potent antagonistic bacterium for many aquatic pathogens, including Vibrio species, is Bacillus pumilus D5. A thorough analysis of the genetic information contained in B. pumilus D5's entire genome led to the discovery of genes involved in the production of antibacterial compounds. High performance liquid chromatography (HPLC) and tandem mass spectrometer (MS/MS) were used to analyze the antibacterial compounds in the supernatant of cultured B. pumilus D5, and an antibacterial test was conducted using the corresponding purified reference materials. B. pumilus D5 has genes that are highly consistent with the three biosynthetic pathways of antibacterial substances, including kanosamine, bacilysin, and toxoflavin, according to a cross-analysis of the whole genome data of the strain with the biosynthetic pathway database of antibacterial substances. The culture supernatant of B. pumilus D5 contained kanosamine, bacilysin, and toxoflavin, according to HPLC and MS/MS analysis. By comparing the peak area of the sample in chromatography with the standard curve created by standard kanosamine and toxoflavin, the concentration of kanosamine (2,800.00 ng/ml) and toxoflavin (1.23 ng/ml) in the culture supernatant was determined, while the content of bacilysin was undetermined because the standard sample for this substance is not available on the market. The antibacterial tests were conducted using the standard kanosamine and toxoflavin and results revealed that toxoflavin can inhibit the growth of Vibrio cholerae, V. harveyi, and V. vulnificus at dose between 25.0-100.0 µg/mL, while 500.0 µg/mL toxoflavin can inhibit the growth of Streptococcus agalactiae and Streptococcus iniae; however, kanosamine has no inhibitory effect on all tested strains up to 5,000.0 µg/mL. Therefore, it is hypothesized that one of the primary antibacterial elements responsible for B. pumilus D5's ability to resist a variety of aquatic pathogens, such as Vibrio spp. and Streptococcus spp., is the toxoflavin it produces.