The aim of this study was to describe the distribution of the trimethoprim-sulfamethoxazole resistance genes and their association with class 1 integrons in a collection of clinical isolates of Enterobacteriaceae recovered at the University Hospital Sahloul in Tunisia. A total of 80 isolates of Enterobacteriaceae were studied, including six different species. There were 35 extended-spectrum beta-lactamases (ESBL)-producing isolates. Resistance to trimethoprim-sulfamethoxazole was assessed by the disk diffusion method. Polymerase chain reaction (PCR) with primers specific for sul1, sul2, and sul3 was used to detect the three known sulphonamide resistance genes. The presence of class 1 integrons in the studied isolates was detected using PCR and the resistance gene cassettes were characterized by directly sequencing the PCR products obtained with 5'conserved segment (5'CS) and 3'conserved segment (3'CS) primers. The int1 gene was found in 68 out of 80 enterobacterial isolates. The sul1 gene was found in 22 isolates (27.5%), sul2 gene in 5 isolates (6.25%), and both genes in 49 isolates (61.25%). Eight of the studied isolates had no dfr alleles, and in the remaining 72 isolates, 7 dfr genes were identified. The most prevalent were dfrA7 (40%) and dfrA17 (33%). Class 1 integrons were found to be an important genetic element of resistance to trimethoprim-sulfamethoxazole among the clinical isolates of Enterobacteriaceae. The types, combinations, and frequency of the gene cassettes in integrons provide useful data for the surveillance of antimicrobial resistance in our hospital and for the prescription practice of cotrimoxazole.