A panel of 223 faecal samples was analysed to determine the clinical utility of the Seeplex® Diarrhea ACE Detection multiplex PCR system (Seeplex system; Seegene, Korea), a qualitative multiplexing PCR technology that enables simultaneous multi-pathogen detection of four viruses and/or ten bacteria associated with acute gastroenteritis. Conventional diagnostic methods and a norovirus-specific multiplex real-time RT–PCR detected 98 pathogens in 96 samples. The Seeplex system detected 81 pathogens in 75 samples. All samples positive for adenovirus, norovirus, Campylobacter spp., Escherichia coli O157, Shigella spp. or Vibrio spp. were detected by the Seeplex system. Rotavirus, Clostridium difficile toxin B, and Salmonella spp. were not detected in 12.5%, 50% and 15.8% of samples, respectively. Additional multiple infections were detected in 19 samples by the Seeplex system. The Seeplex system provides significant additional diagnostic capability for the syndromic diagnosis of acute gastroenteritis with increased sensitivity for the majority of pathogens.