The prevalence of (MDRB) is increasing worldwide; therefore, this study aimed to identify the most common MDRB in clinical specimens and meet the urgentneed to develop new antibacterial drugs to control their intractable infection. Additionally, due to the confrontation of the infection associated with bacterial biofilms, which are difficult to treat, and cause problems to public health, which require real solutions. Bee Venom produced by the glands of (Apis mellifera) is a complex mixture of active peptides, enzymes, and amines. So, it is considered a fertile environment for research to achieve the goal of this study. The results of the specimen’s examination showed that, from a total of 500 clinical specimens, there are 224 specimens exhibited no growth, while 276 were positive. From 276 positive cultures, 317 isolates were obtained. Out of the 317 bacterial isolates, 169 (53.3%) were Gram-negative bacteria (GNB), and 148 (46.7%) were Gram-positive (GP). It was of this number 124 (39.1%) were multidrug-resistant (MDR) isolates of which 89 (71.77%) were Gram-negative type, including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa and 35 (28.23%) were Gram-positive including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Vancomycin-resistant Staphylococcus aureus, Staphylococcus haemolyticus and Enterococcus faecalis. Antibacterial assays showed that Bee Venom possesses strong potential effect against MDR isolates including both GNB and GPB.with a wide range of MICs and MLCs concentration-spacing between 3.125 – 50 μg/mL and 6.25 – 100 μg/mL, respectively against all MDR-GNB and GPB. It was found that GPB was more sensitive at lower concentrations of Bee Venom than GNB. In addition, Bee Venom sub-MICs values against the most biofilm bacterial produces namely; E. faecalis, P. aeruginosa, S. aureus, VRSA, S. haemolyticus and E. faecalis exhibited sharp reduction in their biofilms ranged between (63.8- 92%) especially at ½ MICs according to each bacterium, exclude E. faecalis biofilm was moderately affected (39%). While, at another tested sub-MICs showed moderate, weak, and no antibiofilm effects.