Aim: Diabetic foot is the clinical term describing a series of diabetes-related lower extremity complications, such as ulceration, infection, and gangrene. This study aimed to prospectively investigate the causative aerobic bacteria in diabetic foot ulcer samples, as well as anaerobic bacteria and fungi, which are thought to play an important role in diabetic foot infections. Additionally, the antimicrobial susceptibility of the causative microorganisms obtained from the cultures will be investigated. Methods: Fifty-nine samples were collected from 50 patients with type 2 diabetes mellitus (DM). The BD Phoenix TM automated system was used for species-level identification of microorganisms and antimicrobial susceptibility testing. The antimicrobial susceptibility of anaerobic microorganisms was tested using the gradient test method. Results: Gram-negative aerobic bacteria were found to be the most common agents (65%) in diabetic foot infections, with 65% being multidrug-resistant. Staphylococcus aureus was identified as the most common cause of monomicrobial infections, with a methicillin-resistant rate (MRSA) of 27%. Vancomycin, teicoplanin, and linezolid were the most effective antibiotics against Gram-positive agents, while ceftolozane/tazobactam, piperacillin/tazobactam, meropenem, ertapenem, colistin, and amikacin were the most effective against Gram-negative agents. Conclusions: The high prevalence of multidrug-resistant Gram-negative agents highlights the importance of prudent antibiotic use. Considering DM is 80% preventable through lifestyle changes, increasing public awareness about healthy living is crucial.