The incidence of antibiotic resistance in pathogenic bacteria has become an alarming clinical and social problem. Therefore, the demand for alternative antimicrobial compounds has increased. In this study, a chemical profile of honey bee (Apis mellifera L.) venom (HBV) has been determined by HPLC and FTIR-ATR spectroscopy, and tested for antibacterial activity, as well as efficiency with regard to conventional antibiotics. The investigated HBV was of high quality with melittin and total protein contents of 70.10 ± 7.01%, and 84.44 ± 3.12 g/100 g, respectively. The purity of HBV was confirmed by FTIR-ATR spectral profiling, which revealed a unique pattern of absorption bands that are characteristic of its major fractions. In addition, HBV showed a broad spectrum of activity against all three tested biomasses of potentially pathogenic Gram-positive and Gram-negative bacteria with MIC values ranging between 12.5 and 200 µg/mL, and MBC between 12.5 and 400 µg/mL. When compared to conventional antibiotics, HBV (400 µg) showed up to 27.8% efficiency of tetracycline (30 µg), 52.2% erythromycin (15 µg), 21.2% ciprofloxacin (5 µg), and 34.6% of ampicillin-sulbactam (20 µg). The overall results demonstrate the therapeutic potential of the analyzed HBV.