We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens. A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance. Improved sensitivity was achieved using the protozoan panels and 16 more mixed infections were observed compared to tests with conventional methods. Using the C. difficile panels, 100% sensitivity was achieved when compared to the gold standard of toxigenic culture. In addition, hypervirulent strains including ribotype 027 could be identified directly from primary sample without the need for ribotyping methods. Bacterial and viral panels detecting Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Listeria monocytogenes, norovirus groups I and II, rotavirus A, astrovirus, sapovirus, rotavirus B, adenovirus and adenovirus 40/41 performed as well as conventional methods, whilst allowing detection in 3 hours from processing to result. Multiplex real time PCR panels with universal sample preparation allow streamlined, rapid diagnosis of gastrointestinal pathogens whilst extending the characterisation of pathogens present in stool samples from affected patients.