Background: Diarrhoeal diseases remain a significant global health burden, particularly in low- and middle-income countries. The ability to identify the specific diarrhoeal disease-causing pathogens rapidly and with an appropriate degree of accuracy is crucial both for patient care and preventing the spread of disease. Stool culture is a classic and not as expedient procedure, and does not manage to detect fastidious pathogens effectively. Real-time PCR, which is a fast, reliable, and accurate method for the identification of a wide range of enteric pathogens, increases diagnostic accuracy and facilitates effective antibiotic prescription. Methods: This study included 276 patients with symptoms of diarrhoea and 138 healthy controls. Stool samples were tested for the presence of major bacterial pathogens implicated in the gut using conventional culture and real-time PCR. The subjective information regarding individual backgrounds and health status was gathered. The use of PCR assays allowed for the selection of pathogen-specific genetic markers (such as those in Shigella, Salmonella, Campylobacter, Clostridium difficile, and Vibrio cholerae). Culture procedures complied with the then-laid guidelines in microbiology procedures. Statistical analysis of the results was performed to determine and compare the sensitivity, specificity, and predictive values of individual methods. Results: Out of 276 diarrhoeal patients, real-time PCR detected pathogens in 23 cases, significantly higher than conventional culture (5 cases). PCR showed markedly higher sensitivity (96%) compared to culture (21%), with both methods exhibiting 100% specificity and PPV. Mixed infections and less common pathogens like Clostridium difficile were only identified via PCR. Antibiotic use significantly declined post-PCR diagnosis. PCR-positive patients had higher rates of blood/mucus in stool, abdominal pain, and fever. Antimicrobial susceptibility patterns revealed resistance to commonly used antibiotics, emphasizing the value of accurate, rapid diagnostics for targeted therapy. Conclusion: Based on results from this study, real-time PCR was more sensitive than traditional methods and especially useful for screening immunocompromised or severely afflicted patients. Application of the real-time PCR in clinical settings is likely to deliver better outcomes in patients due to the possibility of making an accurate and timely diagnosis and choosing the appropriate antimicrobial therapy.