<p>Fluoroquinolone-resistant bacteria (FQRB) and their related resistance genes in aquatic ecosystems have become a subject of increasing concern. However, the distribution of FQRB in the Bagmati River and its tributaries in Kathmandu, Nepal remains largely unexplored. This study aimed to analyze the phenotypic and genotypic traits of fluoroquinolone-resistant Enterobacteriaceae isolated from these rivers between March and August 2023. The physico-chemical properties of the river water were assessed, and FQRB present in the rivers were quantified and identified. The disc diffusion method was employed to assess the susceptibility of bacterial isolates to eight commonly used antibiotics. Polymerase chain reaction (PCR) was used to detect fluoroquinolone resistance genes, <i>qnrA</i>, <i>qnrS</i>, and <i>aac-(6')-Ib-cr</i>. The rivers exhibited significantly high levels of turbidity, total suspended solids, and chemical oxygen demand. On average, 43.21% of the bacterial population exhibited resistance to fluoroquinolones, with the highest concentration of FQRB recorded in the Bagmati River (2.62 × 10⁵ ± 0.39 × 10⁵&#xa0;CFU/mL). Among the 210 FQRB isolates, <i>E. coli</i> (40.95%) and <i>K. pneumoniae</i> (21.43%) were the most prevalent species, predominantly identified in the Bagmati River. Antibiotic susceptibility testing revealed that the rate of piperacillin resistance was the highest (100%), followed by ceftazidime (78.10%) and cefepime (75.71%). Additionally, 90.95% of the FQRB exhibited multidrug resistance. PCR analysis identified the resistance genes <i>qnrA</i>, <i>qnrS</i>, and <i>aac-(6′)-Ib-cr</i> in 5.71%, 35.71%, and 55.24% of FQRB, respectively. These findings indicate that river water may act as a substantial reservoir of transmissible antibiotic-resistant bacteria and antibiotic-resistance genes in the Kathmandu Valley, potentially posing serious risks to the health of the local population.</p>