Objective: In the present study, the biological activity of the hexane extract of the honey bee Apis mellifera was evaluated against different pathogenic bacteria, as an alternative to synthetic antibiotics to prevent side effects and bacterial resistance. Methods of Work: Bacterial samples were collected from medical laboratories and outpatient clinics in the city of Tikrit; eight isolates of pathogenic bacteria previously identified using the VITEK system were collected, comprising K. pneumoniae, P.auroginosa, P.fluorescence, Staph. haemolyticus , E. aerogense , S. aureus , P. vulgaris , E. coli . Samples of the honeybee Apis mellifera were collected from the Tarmiyah area, north of Baghdad Governorate, using standard insect collection methods. Extracts were prepared at four concentrations (25, 50, 100, and 200 mg/mL) using hexane as the solvent. The tests were conducted using the agar well diffusion method. Results: The results showed that the 200 mg/ml concentration exhibited the highest inhibitory efficacy, with a mean inhibition zone diameter of 17.1 mm, significantly outperforming the other concentrations, while the lowest concentration (25 mg/ml) showed the least efficacy, with a mean inhibition zone diameter of 9.7 mm. The Gram-positive bacterium S. haemolyticus demonstrated the highest sensitivity, with an inhibition zone diameter of 18.0 mm. In contrast, E. coli (Gram-negative) exhibited the weakest response, with an inhibition zone diameter of 8.25 mm. The inhibitory activity of the extract against the studied bacterial isolates, including multidrug-resistant (MDR) strains, is attributed to the specific biochemical composition of the honeybee extract. The superior performance of the hexane extract is attributed to its high capacity for extracting non-polar, hydrophobic organic compounds such as free fatty acids, their esters, and lipophilic substances, which possess a strong ability to penetrate the lipid layers of microbial cell membranes; this highlights the importance of solvent selection in determining the extract's biological activity.