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The study determined the prevalence, antimicrobial resistant (AMR) determinants, and genetic characteristics of Escherichia coli and Klebsiella pneumoniae isolates from patients with diabetic foot infection (DFI) in a Tunisian hospital. A total of 26 Escherichia spp. and Klebsiella spp. isolates were recovered and identified by MALDI-TOF-MS. Antimicrobial susceptibility testing, the detection of AMR determinants and Shiga-like toxin genes, phylogenetic grouping, and molecular typing were performed. Twelve E. coli , 10 K. pneumoniae , 3 K. oxytoca , and 1 E. hermanii were isolated. A multidrug-resistant phenotype was detected in 65.4% of the isolates. About 30.8% of isolates were extended-spectrum β-lactamase (ESBL) producers and mainly carried bla CTX -M-15 and bla CTX -M-14 genes. One bla NDM -1 -producing K. pneumoniae- ST1 strain was identified. Class 1 integrons were detected in 11 isolates and 5 gene cassette arrangements were noted: dfrA1+aadA1 ( n = 1), dfrA12+aadA2 ( n = 3), and dfrA17+aadA5 ( n = 1). Other non-β-lactam resistance genes detected were as follows (number of isolates): aac(3')-II (3), aac(6')-Ib-cr (8), qnrB (2), qnrS (4), cmlA (2), floR (4), sul1 (11), sul2 (11), and sul3 (2). The phylogroup B1 was the most frequent (41.7%) among E. coli , and two ESBL-producing isolates corresponded to the ST131-B2 lineage. The ESBL- and carbapenemase-producing Enterobacteriaceae in DFIs are described for the first time in Tunisia.