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Antibiotic-resistant bacteria continue to be of major health concern world-wide. Thus, it is of great interest to study the biological properties and determine active compounds in natural products likely to be used as new health remedies. Therefore, the main objective of this work is to test the antibacterial activity of royal jelly samples, defatted royal jelly samples and their ethyl ether extracts against bacteria capable of infecting cutaneous wounds in humans and animals, and to recognize major bioactive compounds by using bioassay directed identification. The microorganisms used in the study were Staphylococcus aureus (including Methicillin-resistant and sensitive strains), Staphylococcus epidermidis, Micrococcus luteus, Streptococcus uberis, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. The activity of royal jelly samples to inhibit bacterial growth was assessed by using well-difussion tests. Direct bioautography was used to identify bioactivity, and uv-visible spectroscopy and gas chromatography-mass spectrometry were used to identify bioactive compounds. Overall, royal jelly samples showed higher growth inhibition activity against Gram positive bacteria as compared to Gram negative bacteria. The growth of bacterial strains belonging to the Enterococcus and Streptococcus genders was less affected by the presence of royal jelly than bacterial strains of the Staphylococcus gender did. Compounds with antibacterial activity were found in the ethyl ether extract of royal jelly samples. 10-hydroxy-2-decenoic acid was the major component identified in the purified fraction obtained by bioassay guided fractionation of the ethyl ether extract. In conclusion, bioactivity of royal jelly samples is mainly due to their 10-hydroxy-2-decenoic acid content.