Diabetic foot ulcer (DFU) is the most serious diabetic complication. Gangrene is causing by the successive bacterial infection invading diabetic wounds and may lead to limb amputation for the diabetic patient. The bacteria inhabiting the wound exhibit the ability to forming biofilm, which is a protected mode of growth that allows cells to survive under harsh environments while also dispersing to colonize new niches. The main objective is to clarify the reasons of forming the bacterial biofilm, as well as, the multidrug resistant bacteria. The swab was used method for sample collection and bacterial isolation; different culture media as (BHI medium, MSA and TSA) were used for getting pure cultures from the pathogenic bacteria invading the diabetic wounds. Crystal violet was applied to detect the biofilm formed by these bacteria. Staphylococcus species are the most prominent bacteria invading the diabetic wounds forming a stable biofilm resistant to many antibiotics, one hundred bacterial isolated were recovered from diabetic foot ulcer belonging to five genus, namely, Staphylococcus sp., Klebsiella sp., E.coli, Pseudomonas sp., and Bacillus sp. using phenotypic analysis was confirmed by phylogenetic analysis.