Abstract Background The use of gastrointestinal disease multiplex polymerase chain reaction (GI PCR) testing has become common for suspected gastrointestinal infection. Patients often test positive for multiple pathogens simultaneously through GI PCR, although the clinical significance of this is uncertain. Methods This retrospective cohort study investigated risk factors and clinical outcomes associated with detection of multiple (as opposed to single) pathogens on GI PCR. We included adult patients who underwent GI PCR testing from 2020 to 2023 and had one or more pathogens detected. We compared patients with multiple versus those with single pathogens and hypothesized that immunosuppression would be a risk factor for detection of multiple pathogens. We further hypothesized that, during the 90 days after GI PCR testing, patients with multiple pathogens would have worse clinical outcomes such as increased rates of ED visits, death, hospitalization, or ambulatory care visits. Results GI PCR was positive in 1,341 (29%) of tested patients; 356 patients had multiple pathogens and 985 had one pathogen. The most common pathogens included Enteropathogenic Escherichia coli (EPEC, 27%), norovirus (17%), and Enteroaggregative E. coli (EAEC,14%) in both multiply and singly positive patients. Immunosuppression was not associated with multiple pathogens (adjusted odds ratio [aOR] 1.35, 95% CI (0.96, 1.86). The factors most associated with multiple pathogens were Hispanic ethnicity (OR 1.86, 95% CI 1.42, 2.45) and chronic kidney disease (OR 1.69, 95% CI 1.13, 2.49). Patients with multiple pathogens were more likely to have ED visits during the 90 days after GI PCR testing (40% vs. 32%, p<0.01), but they were not more likely to die, be hospitalized, or to have ambulatory medical visits. Conclusions Immunosuppression was not associated with detection of multiple as opposed to single pathogens on GI PCR testing. There were worse clinical outcomes associated with detection of multiple pathogens, although these effects were modest.