We have evaluated the multiplex molecular method xTAG® Gastrointestinal Panel (GPP) for detecting pathogens in stool samples of diarrhoeic patients. We collected 440 samples from 329 patients (male:female ratio of 1.2:1), including 102 immunosuppressed adults, 50 immunosuppressed children, 56 children attending the neonatal unit and 121 children attending the emergency unit. Of these, 176 samples from 162 patients were xTAG® GPP positive (102 viruses, 61 bacteria and 13 parasites) and the assay was more sensitive than the conventional test for detecting rotavirus (p <0.01), noroviruses (p <0.0001), Salmonella spp. (p <0.001), Campylobacter spp. (p <0.001) and toxigenic Clostridium difficile (p 0.005). The predominant pathogens were viruses (23.2%), with rotavirus (15.9%) being the most common. Bacterial agents were detected in 13.9%; the most common was Salmonella spp. (4.8%). Parasites were detected in 2.9%; Cryptosporidium spp. (2%) was the most common. There were 31 co-infections (7% of samples), involving two pathogens in 23 (5.2%) and three pathogens in eight (1.8%) samples. There were 113 (92.6%) positive samples from the children attending the emergency unit, 25 (17%) positive samples from immunosuppressed adults, 22 (25.3%) positive samples from immunosuppressed children and 16 (19%) positive samples from children attending the neonatal unit. The low turnaround time and technical hands-on time make this multiplex technique convenient for routine use. Nevertheless, conventional bacterial culture and parasitological stool examination are still required to detect other pathogens in specific cases and to determine susceptibility to antibiotics.