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According to recent studies, there are almost 435 million people worldwide with diabetes mellitus. It is estimated that of these 148 million will develop Diabetic foot ulcers (DFUs) during their lifetime, of which 35 to 50% will be infected. In this scenario, the presence and frequency of pathogenic microorganisms and their level of susceptibility to the most frequent classes of antibiotics used to treat this pathological condition from patients with DFUs admitted to the outpatient clinic of vascular surgery of the Federico II University Hospital of Naples from January 2019 to March 2021 were investigated. Furthermore, the diabetic population characteristics under study (i.e., general, clinical, and comorbidities) and the pathogenic bacteria isolated from lesions were also considered. Bacterial strains poorly susceptible to antibiotics were more frequent in polymicrobial infections than in monomicrobial infections. β-Lactams showed the highest levels of resistance, followed by fluoroquinolones, aminoglycosides, and finally macrolides. The main findings of the study demonstrated that the occurrence of resistant microorganisms is the dominant factor in ulcer healing; thus it is essential to investigate the antibiotics' susceptibility before setting antibiotic therapy to avoid inappropriate prescriptions that would affect the treatment and increase the development and spread of antibiotic resistance. [ABSTRACT FROM AUTHOR]