Background: Urinary tract infections represent a major expensive, common public health problem worldwide due to their high prevalence and the difficulties associated with their management. Objectives: This study aimed to characterize the Enterobacter cloacae strains isolated from urinary tract infections in the medical diagnostic laboratories of Shahrekord, Iran. Methods: Urine samples from patients with urinary tract infections from the Shahrekord medical diagnostic laboratories located in Chaharmahal and Bakhtiari Province, Iran, were collected from June 2019 to February 2020. When the samples were cultured, the different isolates of E. cloacae were identified by biochemical tests. Biofilm production capacity was evaluated. Bacterial susceptibility to antibiotics was determined using the Kirby Bauer method, and antibiotic resistance genes were researched by the multiplex PCR technique. Results: In this study, 65 isolates of E. cloacae were obtained. The highest percentage of resistance was observed for co-trimoxazole (84.62%), ampicillin (76.93%), tetracycline (73.85%), and above half of the E. cloacae strain isolates (53,85%) were strongly involved in biofilm production. Some genes, including qnr A, qnr B, qnr S, tetA, tet B, sul1, bla CTXM, bla SHV, and(2)la, ant(3)la, and aac(3)IIa, were detected in the genome of these isolates. Conclusions: The strains are multi-resistant, and their resistance has already reached the carbapenem class. This requires further investigation, and urgent measures must be adopted.