Background: Diabetic foot infection (DFI) is considered the most common cause of diabetes-related hospitalization. Diabetic foot ulcers are subjected to bacterial colonization with biofilm forming organisms which are difficult to eradicate. The aim of this study was to identify the spectrum of bacteria associated with DFI and their ability to form biofilm, to evaluate differences in antibiotic susceptibility pattern between planktonic and biofilm phases, and to determine the antibiotics which are active on the organism in the biofilm phase. Methods: The study comprised 50 patients with DFI. A deep swab was collected from each patient and cultured. All isolates were identified and screened for biofilm formation. Biofilm forming isolates were further subjected to minimum biofilm eradication concentration (MBEC) assays to determine resistance to different antimicrobials while in the biofilm phase. Results: Seventy-one isolates were identified, (14.1%) were Gram positive cocci, (83.1%) were Gram negative bacilli, and (2.8%) were Candida species. The most frequently isolated organism was Klebsiella spp. (18/71, 25.4%), followed by Proteus spp. (14/71, 19.7%). The prevalence of biofilm forming isolates was 38%. All the studied isolates showed MBEC higher than the MIC for all tested antimicrobials. Conclusions: The substantial discrepancy between MIC and MBEC results observed in this study emphasizes the lack of reliability of the routine antimicrobial susceptibility testing in case of biofilm formation. Among all tested antimicrobials; cefoperazone/sulbactam, gentamicin, and vancomycin demonstrated activity against bacteria in the biofilm phase.