Bee venom (BV), or apitoxin, is a complex substance produced by a gland in the abdominal cavity of bees. The main component of BV is melittin, which is a largely studied substance due to its biological properties. To date, the most well-known bee venom and melittin are derived from domesticated honey bees, while venom and melittin derived from wild honey bees have been under-investigated. Hence, this study primarily reports the antimicrobial activities of bee venom and synthetic melittin derived from four different honey bee species (Apis mellifera, A. cerana, A. dorsata, and A. florea) in Thailand. All the bee venom extracts and melittins showed more robust antibacterial activities against Gram-positive (Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, S. aureus MRSA, and S. epidermidis) than Gram-negative bacteria (Escherichia coli, Klebsiella pneuminiae, and Salmonella typhimurium) or a fungus (Candida albicans), while the synthetic melittins also have antimicrobial activity at higher concentrations than the bee venom extract. Furthermore, the A. cerana venom extract showed the highest activity against the tested bacteria, followed by A. mellifera, A. florea, and A. dorsata. Therefore, A. cerana venom may be further developed for use in medical applications as a potential alternative agent against Gram-positive bacteria and antibiotic-resistant bacteria.