This study presents a comprehensive investigation into the microbial ecology of diabetic foot infections (DFIs), using molecular-polymerase chain reaction (PCR) analysis to accurately identify the causative agents. One hundred DFI patients were recruited and classified using the Depth Extent Phase and Associated Etiology (DEPA) score according to their severity. Results revealed polymicrobial infections in 75% of cases, predominantly featuring (83%) and (63%). Importantly, 20% of samples exhibited facultative anaerobes or , exclusively in high DEPA score ulcers. coinfection was identified in 19.2% of cases, underscoring the need for mycological evaluation. Empirical antimicrobial therapy regimens were tailored to DEPA severity, yet our findings highlighted a potential gap in methicillin-resistant (MRSA) coverage. Despite an 88% prevalence of methicillin-resistant , vancomycin usage was suboptimal. This raises concerns about the underestimation of MRSA risk and the need for tailored antibiotic guidelines. Our study demonstrates the efficacy of molecular-PCR analysis in identifying diverse microbial communities in DFIs, influencing targeted antibiotic choices. The results advocate for refined antimicrobial guidelines, considering regional variations in microbial patterns and judiciously addressing multidrug-resistant strains. This research contributes crucial insights for optimizing DFIs management and helps the physicians to have a fast decision in selection the suitable antibiotic for each patient and to decrease the risk of bacterial resistance from the improper use of broad-spectrum empirical therapies.