To characterize microbial distribution and antimicrobial susceptibility profiles in purulent specimens from pediatric acute appendicitis, guiding evidence-based antibiotic therapy optimization. In this prospective cohort study, intraoperative pus samples were collected from 70 consecutive pediatric patients undergoing appendectomy between January 2024 and January 2025. Bacterial identification was performed using the VITEK 2 Compact system. Antimicrobial susceptibility testing was conducted via broth microdilution for minimum inhibitory concentration (MIC) determination. Extended-spectrum β-lactamase (ESBL) production was confirmed using the Clinical and Laboratory Standards Institute (CLSI) double-disk synergy test. Pathogens were isolated from 88.6% of specimens (62/70), with Gram-negative bacteria predominating (98.3%, 61/62). The most common isolates were Escherichia coli (74.2%, 46/62) and Pseudomonas aeruginosa (14.5%, 9/62). E. coli exhibited high susceptibility to amikacin (93.5%), imipenem (97.8%), and tigecycline (100%), but elevated resistance to co-trimoxazole (82.6%) and ceftriaxone (78.3%). All P. aeruginosa isolates were susceptible to the tested antimicrobial agents. ESBL production was detected in 41.3% (19/46) of E. coli isolates. Gram-negative pathogens, particularly E. coli and P. aeruginosa, are the primary etiological agents in pediatric acute appendicitis. Empirical antibiotic therapy should prioritize agents effective against ESBL-producing strains, with carbapenems or aminoglycosides recommended as first-line options pending local susceptibility data.