Given antibiotic resistance in pathogens, finding antibiotics from new sources is always a topic of interest to scientists. In the present study, among various isolates from the Persian Gulf coastal area, the strain RP137 was selected as producer of antibacterial compound. Morphological and biochemical studies along with 16S rDNA sequencing showed that strain RP137 belongs to Bacillus genus and was tentatively named Bacillus velezensis strain RP137. The effect of various carbon and nitrogen sources on optimizing the production of antibacterial compound showed that the low-cost rice starch and potassium nitrate supply to the strain RP137 caused producing of 86.08.7 mu g/mL extract having the antibacterial activity. The fractionation of the primary methanol extract in different solvents followed by reversed-phase HPLC obtained a pure antibacterial-active sample, S-137-R. Structural analysis of the purified S-137-R with the help of FTIR, HR-MS, H-1-NMR, and C-13-NMR showed that the S-137-R compound is classified as aminoglycoside. Minimum inhibition concentration (MIC) of the pure compound for Gram-positive bacteria, Staphylococcus aureus and methicillin resistant Staphylococcus aureus, showed an average antibacterial effect of about 80 mu g/mL and 150 mu g/mL, respectively and for Pseudomonas aeruginosa (100 mu g/mL), while having very little toxic effect on E. coli. Moreover, low cytotoxicity effect of the S-137-R on cancerous and normal cells as well as the low intensity of the hemolysis of red blood cells in higher concentrations of S-137-R make it an ideal candidate for further structure-activity relationship assessments towards its medical applications. [GRAPHICS] .