Acute gastrointestinal infections (AGIs) can lead to significant morbidity and mortality. In diagnosing AGI, culture-independent diagnostic tests offer advantages over traditional methods and increase the chance of detecting multiple pathogens (co-detection). A retrospective analysis of data from a tertiary pediatric hospital was conducted to characterize occurrence of AGI co-detections and compare outcomes with patients who had only one AGI pathogen detected. Medical records were obtained for patients with stool samples tested using BioFire FilmArray GI Panel between 1 January 2016 and 31 December 2020. Data were described using descriptive statistics, correlation analysis, and logistic regression to identify risk factors and estimate co-detection rates. During the study period, 12,753 patients had a total of 17,159 stool samples tested. Of these, 8,212(47.9%) tested positive, with 6,040(73.6%) being single detections and 2,172(26.4%) being co-detections. Patients with single detection experienced higher hospitalization rates than patients with co-detection. Patients 1–4 years old exhibited the highest co-detection rate relative to other age groups, while Hispanic/Latino individuals were 1.75 times more likely to have co-detection than other races. This study emphasizes the significance of understanding pathogen interactions concerning clinical characteristics and epidemiology of AGI, and the necessity for effective diagnostic strategies and optimal healthcare resource allocation.