The larvae of the black soldier fly (BSFL), Hermetia illucens (Linnaeus, 1758) (Diptera: Stratiomyidae), can survive in environments contaminated with various bacteria by producing antimicrobial compounds. This study, for the very first time, investigated the potential antibacterial activity of hemolymph extracted from BSFL in Malaysia using diffusion and dilution methods. Prior to extraction, the larvae were infected with either Methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli. Then, the hemolymph was collected. Serial dilutions from 200 to 12.5 mg/ml of the hemolymph extracts were screened against ten different bacteria. The results showed inhibition of eight out of ten tested bacteria (i.e., MRSA, Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Klebsiella pneumoniae and Acinetobacter sp.). We found that immunological-challenge larvae have stronger antimicrobial activity than the control groups. The Minimum Inhibitory Concentrations (MIC) for bacteria against for infected larvae were 12.5 mg/ml for MRSA, S. pyogenes, B. subtilis, M. luteus, E. coli, and K. pneumoniae. As for bactericidal activity, the MBC of E. coli infected larvae was 25mg/ml against S. pyogenes and B. subtilis. In conclusion, BSFL hemolymph has antibacterial activity against a range of bacteria and could be a candidate for novel antimicrobial development.