We sought to define the type and antibiotic sensitivity response of pathogens isolated from diabetic foot ulcers based on ulcer location and Wagner classification. This retrospective analysis was done among 126 patients between 40 and 70 years of age at the Prince Sultan Military Medical City, Riyadh, Saudi Arabia, using the swab culture method for microbiological assessment. Data on demographic variables, ulcer sites (location), and depth of ulcer (Wagner classification) were also collected. A total of 134 pathogens were isolated from 126 patients. The most common gram-negative pathogen identified was Pseudomonas aeruginosa (15.6%), followed by Klebsiella spp (6.7%). The most common gram-positive pathogen was Staphylococcus aureus (35%), followed by Streptococcus (8.9%). The most widely accepted antibiotic to which the gram-negative bacteria were sensitive was gentamycin (20.1%), followed by ciprofloxacin (19%). The most routinely used antibiotic in the treatment of the gram-positive bacteria was erythromycin (16%), with trimethoprim-sulfamethoxazole (14.1%) being the next most effective. In conclusion, Pseudomonas aeruginosa, Klebsiella spp, Staphylococcus aureus, and Streptococcus were the most common causes of diabetic foot infections. However, the frequency of encountered pathogens and respective sensitivity to antibiotic therapy may be influenced by the location site and severity of the ulceration.