Marine actinomycetes are promising sources of antibacterial compounds that may alleviate antimicrobial resistance. This study aimed to evaluate the antibacterial potential of marine actinomycetes secondary metabolites against staphylococci and to characterize their mechanism of action. The actinomycetes were obtained from Salt Rock Beach, South Africa using actinomycetes isolation agar medium and identified by 16S rRNA sequencing. Antibacterial efficacy was evaluated using broth microdilution method. The effect of the secondary metabolites on the respiratory chain dehydrogenase activity and membrane integrity of staphylococci was analyzed by iodonitrotetrazolium chloride and 260 absorbing materials assays, respectively. Liquid chromatography-mass spectrometry analysis was done to identify the chemical constituents of the bacterial extract. Streptomyces SRST1SA2 was the only isolate whose secondary metabolites revealed anti-Staphylococcus activity. The extract from Streptomyces SRST1SA2 showed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranging from 0.25 to 1 mg/ml and 0.5–1 mg/ml, respectively. The extract inhibited bacterial growth by interfering with the activity of respiratory chain dehydrogenase and membrane integrity. The LC–MS spectrum revealed antibacterial compounds such as myristic acid (C₁₄H₂₈O₂), palmitic acid (C₁₆H₃₂O₂), and oleic acid (C₁₈H₃₄O₂). Streptomyces sp. SRST1SA2 has the potential to serve as a source of antibacterial compounds against staphylococci.