Background The biofilm phenomenon represents a prevalent mode of microbial life in nature which is characterized by cells irreversibly attaching to surfaces or each other and getting embedded in a matrix of extracellular polymeric substances. Object This study aimed to identify and characterize the genes associated with the common bacterial species responsible for biofilm formation in the catheters of hospitalized patients. Method Different bacterial strains were collected from catheterised patients at three local Jordanian hospitals for biofilm formation. The isolates were identified using Gram stain and Remel Rapid test. Biofilm formation was detected using the Tube method and tissue culture plate method. Result The presence of fimA and csgD genes was detected by polymerase chain reaction (PCR). Gram-negative bacteria species were isolated on the urethral catheters and the result shows the majority of the isolates were E. coli (40%), followed by K. pneumonia (27%). In comparison, the least was Citrobacter sp (2.6%). Similarly, some Gram-positive bacteria were also identified such as Staphylococcus aureus (4%) and Staphylococcus epidermidis (2.6%). K. pneumonia is strongly associated with biofilm formation (45%) followed by E. coli (33%). Conclusion Biofilm-associated genes, fimA and csgD were detected in all biofilm-positive samples containing the F plasmid, while csgD was detected in all biofilm-negative samples. Biofilm formation tends to be a prevalent process in patients included in this study and may cause dangerous complications in the patients especially in the elderly due to prolonged catheterization periods.