Introduction: The most prevalent infections in outpatient and inpatient settings are urinary tract infections (UTIs), which can be treated with a variety of antibiotics. Fluoroquinolones are the preferred antibiotic class and Prulifloxacin is one of these agents. It can be tested in vitro for its activity against urinary isolates of the bacteria Escherichia coli(E. coli) and Klebsiella pneumoniae (K. pneumoniae) for the purpose of surveillance, along with other fluoroquinolones. Objective: Evaluation of the fluoroquinolone susceptibility of urine isolates of K. pneumoniae and E. coli with a focus on Prulifloxacin. Results: 877 urine samples processed, 257 showed positive cultures, satisfying the Kass concept/Significant bacteriuria. Of these, 106 (41.2%) contained E. coli and K. pneumoniae, of which 92 (87%) were E. coli and 14 (13%) were K. pneumoniae. E. coli exhibited the highest susceptibility to Levofloxacin (61.9%), followed by Prulifloxacin (58.6%) where as to K. pneumoniae Levofloxacin (42.8%) and Prulifloxacin (50%). Conclusion: Levofloxacin is thought to be a drug reserved for the treatment of multi-drug resistant tuberculosis (MDR-TB), the study provides insight into the susceptibility patterns of urinary isolates of E. coli and K. pneumoniae against Prulifloxacin, which can be preferred as the drug of choice for the treatment of UTIs.