Due to its great medical and pharmaceutical importance, honey bee venom is considered to be well characterized both chemically and in terms of biomedical activity. However, this study shows that our knowledge of the composition and antimicrobial properties ofApis melliferavenom is incomplete. In this work, the composition of volatile and extractive components of dry and fresh bee venom (BV) was determined by GC-MS, as well as antimicrobial activity against seven types of pathogenic microorganisms. One-hundred and forty-nine organic C1–C19compounds of different classes were found in the volatile secretions of the studied BV samples. One-hundred and fifty-two organic C2–C36compounds were registered in ether extracts, and 201 compounds were identified in methanol extracts. More than half of these compounds are new to BV. In microbiological tests involving four species of pathogenic Gram-positive and two species of Gram-negative bacteria, as well as one species of pathogenic fungi, the values of the minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) were determined for samples of dry BV, as well as ether and methanol extracts from it. Gram-positive bacteria show the greatest sensitivity to the action of all tested drugs. The minimum MIC values for Gram-positive bacteria in the range of 0.12–7.63 ng mL−1were recorded for whole BV, while for the methanol extract they were 0.49–125 ng mL−1. The ether extracts had a weaker effect on the tested bacteria (MIC values 31.25–500 ng mL−1). Interestingly,Escherichia coliwas more sensitive (MIC 7.63–500 ng mL−1) to the action of bee venom compared toPseudomonas aeruginosa(MIC ≥ 500 ng mL−1). The results of the tests carried out indicate that the antimicrobial effect of BV is associated with the presence of not only peptides, such as melittin, but also low molecular weight metabolites.