Background: Pathogenic Escherichia coli (PEC) is a leading cause of acute diarrhea worldwide. With increasing antibiotic use, antimicrobial resistance in PEC has become a significant public health concern. In this study, we investigated the distribution and antimicrobial susceptibility patterns of PEC isolated from patients with acute diarrhea in the Busan and Gyeongnam regions of South Korea. Methods: In this retrospective cross-sectional laboratory-based study, 272 PCR-positive residual stool or rectal swab specimens collected between October 2023 and June 2024 were screened; after exclusion of 80 specimens without recoverable or confirmable isolates, 192 pathogenic E. coli isolates were included for pathotype identification and antimicrobial susceptibility testing. Pathotypes were identified using multiplex real-time polymerase chain reaction, and antimicrobial susceptibility was tested against 18 agents using the VITEK II system. Results: Enteroaggregative E. coli (EAEC) was the most prevalent pathotype (44.8%), followed by enteropathogenic E. coli (34.9%). High resistance rates were observed for ampicillin (77.1%), cefazolin (55.2%), and amoxicillin/clavulanic acid (44.3%). EAEC demonstrated the highest multidrug resistance (MDR) rate (34.9%). Pediatric patients (< 18 years) exhibited significantly higher resistance rates and MDR frequencies compared with adults, particularly against β-lactam antibiotics. Conclusion EAEC was identified as the predominant and most resistant pathotype in this region. The elevated resistance levels in children and the prevalence of MDR EAEC underscore the need for continuous local surveillance and appropriate antibiotic stewardship.