The resistance to antibiotics in Gram-negative bacilli causing sepsis is a warning sign of failure of therapy. Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) represent major Gram-negative bacilli associated with sepsis. Quinolone resistance is an emerging resistance among E. coli and K. pneumoniae. Therefore, the present study aimed to study the presence of plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, and qnrS by polymerase chain reaction (PCR) in E. coli and K. pneumoniae isolated from pediatric patients with sepsis. This was a retrospective cross-sectional study that included pediatric patients with healthcare-associated sepsis. The E. coli and K. pneumoniae isolates were identified by microbiological methods. PMQR genes namely qnrA, qnrB, and qnrS were detected in E. coli and K. pneumoniae isolates by PCR. The results were analyzed by SPPS24, and the qualitative data was analyzed as numbers and percentages and comparison was performed by Chi-square test, P was significant if < 0.05. The most prevalent gene detected by PCR was qnrA (75%), followed by qnrB (28.1%), and qnrS (25%). The most frequently detected qnr gene in E coli and K. pneumoniae was qnrA (28.8%, and 16.3% respectively). The present study highlights the high prevalence of ciprofloxacin resistance among E. coli and K. pneumoniae isolated from pediatric patients with healthcare-associated sepsis. There was a high frequency of PMQR genes in E. coli and K. pneumoniae isolated from pediatric patients. Therefore, it is important to monitor the spread of PMQR genes in clinical isolates to ensure efficient antibiotic use in those children. The finding denotes the importance of an antibiotics surveillance program.