Diabetic ulceration is a multi-factorial problem which is responsible for considerable morbidity threatening the health care system. By knowing the clinical profile and bio-burden on diabetic ulcer, it is highly beneficial for health treatment. The purpose of the present study was to evaluate the diversity of major bacterial etiology in diabetic ulcer patients. The different samples like pus, swab, and infected tissues were collected from diabetic ulcer patients aseptically and samples were transported through cold chain to the laboratory. The samples were cultured in nutrient agar, mannitol salt agar, macConkey agar and blood agar. Suspected colonies were biochemically confirmed for the isolation of Staphylococcus aureus, Pseudomonas aeruginosa, E.coli and Klebsiella spp. In total 150 diabetic ulcer patients suffering from diabetic foot ulcers, gangrene, burn cases and accidental ulcer cases were analyzed. However, most of the patients developed mono-microbial infection; S.aureus was the most prevalent microbe in diabetic ulcer cases, which were positive for nucA gene.