Background: Diabetic foot ulcer (DFU) is an open sore or wound on the foot of a person with diabetes, and is most commonly located on the plantar surface, or bottom of the foot. The defect is relatively common in adult and elders with diabetes mellitus. Infected lower extremities wound with multi-drug resistant bacteria usually are associated with increased morbidity, mortality and long-term disabilities among diabetic patients. Although the burden of DFU is known in Tanzania (and Mwanza in particular), there is limited information on the patterns of pathogens associated with DFU in our setting, on bacterial and fungal pathogens which in turn limit specific management options to these patients. Methodology: Analytical cross section hospital-based study was conducted among patients with DFU admitted or attending outpatient clinics at BMC from May to July 2022. A structured questionnaire was used to collect socio-demographic, clinical and laboratory data from patients with DFU. Tissue culture was obtained from the base of the ulcer after cleaning with normal saline followed by removing of cellular debris and normal flora over the lesion. Finally, the obtained samples were subjected to culture methods to identify the presence of pathogens (bacteria and fungi) and antimicrobial susceptibility profiles of bacteria. Result: During the study period and based on inclusion criteria, a total of 71 patients, with a mean age of 59.2 ± 13.0 years were recruited. The majority of the participants were male 47 (66.2%). A total of 60 (84.5%) samples were culture positive, resulting into a total of 92 microorganisms isolated. More than half of cultures 34 (56.7%) revealed the presence of single microorganisms. Pathogens isolated were both bacterial 70 (76.1%) and fungi species 22 (23.9%), all bacterial isolates were aerobic. P. aeruginosa and E. coli were most frequent isolated gram-negative bacteria 12(23.5%) and 9(17.6%) respectively. On other hand common isolated gram-positive strains were S. aureus 13 (68.4%) out of all 19 gram-positive culture isolates. A total of 22 fungi spp were isolated, among them 7 (31.8%) were yeast C. albicans 3 (42.8%), A. fumigatus were frequent isolated 13 (86.7%). Polymicrobial growth was observed in 43.3% samples against 56.7% of monomicrobial growth. Most prevalent gram-negative bacteria Pseudomonas aeruginosa showed low resistance to ciprofloxacin, meropenem, gentamicin, piperacillin tazobactam (7.1%, 21.4%, 21.4% and 21.4 respectively) but more resistant to cephalosporins. Staphylococcus aureus showed low resistance to ciprofloxacin, gentamicin and clindamycin (30.8%, 15.4%, and 38.5% respectively), Total MDR bacteria isolates were 64.3%, where methicillin resistance staphylococcus aureus (MRSA) were 76.9%. More than 85.7% of the patient with higher grade ulcer, stage 3 and 4 according to Wagner’s classification were positive to diabetic foot infection and 14.3% of patients with Wagner’s stage 3 and 4 were not infected on their ulcers. Conclusion: Gram negative bacteria were most commonly isolated than gram positive bacteria in causing DFU infections. For gram negative spp the most effective antibiotic were ciprofloxacin, gentamicin, piperaccilin tazobactam and meropenem. Third generation cephalosporins, amoxicillin clavulanate showed poor effectiveness. All cases of DFU infection should therefore be subjected to culture and antimicrobial sensitivity testing for targeted infection management. More studies involving anaerobic pathogens and antifungal susceptibility patterns recommended.