Gastrointestinal (GI) infections are a major health problem all over the world, causing an increase in hospitalization, morbidity, and mortality. The etiological agents of infectious gastroenteritis are viruses, bacteria, and parasites. A precise identification of GI pathogens is crucial for proper treatment and/or isolation, management, and further investigations like designing specific prevention modalities, vaccination strategies, and empiric treatment regimens to prevent the spread of the infectious agents. Routinely, the laboratory diagnosis of GI infections depends on microscopy, culture, and antigen detection. The drawbacks of conventional method are its low sensitivity and 3–5 days of turnaround time in the finalization of report. Quick turnaround time is of paramount value in diagnosis, clinical management, and infection control. From the last decade, molecular-based diagnostic tools have emerged for GI infections in the microbiological laboratory analyses. Culture-independent diagnostic tests typically involve nucleic acid amplification of the genetic material of several bacteria, viruses, and parasites simultaneously. Even whole-genome next-generation sequencing is important for symptomatic patients that remain negative by both routine and multiplex PCR-based diagnostic methods. Therefore, the use of proficient methods for pathogen detection is necessary to ensure prompt turnaround time. This review includes various conventional and molecular tools in identifying various enteropathogens and also analyzes the advantages and drawbacks of all methods.