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Background: Diabetic foot ulcer (DFU) is a major complication of diabetes mellitus (DM) which is associated with high morbidity and mortality. There is high rate of bacteria colonization especially in those with tendencies for poor wound dressing. This is accompanied by high rate of inappropriate antibiotic usage. The aim of this study is to characterize microbial pathogens colonizing foot ulcers of diabetic patients in Enugu, Nigeria, and to determine the antibiotic susceptibility of these isolates. Methodology: This was a descriptive cross-sectional study of consecutively enrolled diabetic patients with foot ulcers in two tertiary healthcare facilities in Enugu, Nigeria, between May 2021 and February 2022. A structured questionnaire was used to obtain socio-demographic and clinical data of the patients. Pus samples and/or tissues were collected from the ulcer lesion of each patient for aerobic and anaerobic microbial cultures and biochemical identification using standard conventional techniques. Results: A total of 310 diabetic patients with foot ulcers were recruited into the study, with 62.3% (193/310) males and 37.7% (117/310) females, and mean age of 56.0±13.9 years. Bacteria and yeast were isolated from samples of 280 (90.3%) patients while samples of 30 (9.7%) patients had no microbial growth. Males had higher frequency of microbial isolates (90.7%, 175/193) than females (89.7%, 105/117), while the age group ≤ 40 years had higher frequency of microbial isolates (100%, 43/43) compared to other age groups, although the differences are not statistically significant (p>0.05). The distribution of the isolates showed that 15.7% (44/280) were monomicrobial while 84.3% (236/280) were polymicrobial. The highest single isolate was Bacteroides fragilis with 5.0% (14/280), followed by Staphylococcus aureus with 3.2% (9/280). Bacteroides fragilis and S. aureus occurred as the highest combined bacteria isolates with 5.7% (16/280). Most of the patients were colonized by combination of bacterial isolates. The susceptibility indicates that most of the anaerobic bacteria were sensitive to metronidazole while S. aureus isolates were resistant to ofloxacin at a rate of 65.0%. Conclusion: The findings in this study showed that there is high bacteria and fungi colonization of foot ulcers of diabetic patients in Enugu, Nigeria. Routine care of wounds especially frequent changes of dressing materials and the use of potent antiseptics, are recommended. [ABSTRACT FROM AUTHOR]