Pediatric diarrhea continues to pose a significant global health challenge, leading to considerable morbidity and mortality among children under the age of five, particularly in low-and middle-income countries. Accurate microbiological analysis is crucial for identifying a range of causative agents, including bacterial, viral, parasitic, and, in some cases, fungal pathogens, which can co-infect tissues and contribute to varying degrees of disease severity. Although conventional diagnostic methods are available, they often suffer from prolonged turnaround times and inconsistent sensitivity. This study aims to assess the effectiveness of advanced molecular diagnostic techniques, specifically multiplex PCR, next-generation sequencing, and whole-genome sequencing, in enhancing pathogen detection and characterizing microbial profiles in cases of pediatric diarrhea. Our findings show that molecular methods significantly improved the detection rates of mixed infections compared to traditional diagnostics. They also allowed us to identify markers of antimicrobial resistance and patterns of microbiome dysbiosis associated with severe disease. Furthermore, the integration of metabolomic and proteomic data provided additional insights into the interactions between hosts and pathogens. © 2026, Editorial board of Journal of Experimental Biology and Agricultural Sciences. All rights reserved.