A multiplex polymerase chain reaction panel facilitated the more rapid institution of appropriate antimicrobial therapy in patients with acute gastroenteritis compared to traditional diagnostic methods. Abstract Background Molecular syndromic diagnostic panels can enhance pathogen identification in the approximately 2-4 billion episodes of acute gastroenteritis that occur annually worldwide. However, the clinical utility of these panels has not been established. Methods We conducted a prospective, multi-center study to investigate the impact of the BioFire FilmArray Gastrointestinal polymerase chain reaction panel on clinical diagnosis and decision-making, and compared the clinical acuity of patients with positive results obtained exclusively with the FilmArray with those detected by conventional stool culture. A total of 1887 consecutive fecal specimens were tested in parallel by FilmArray and stool culture. Laboratory and medical records were reviewed to determine rates of detection, turnaround times, clinical features, and the nature and timing of clinical decisions. Results FilmArray detected pathogens in 35.3% of specimens, compared to 6.0% for culture. Median time from collection to result was 18 hours for FilmArray and 47 hours for culture. Median time from collection to initiation of antimicrobial therapy was 22 hours for FilmArray and 72 hours for culture. Patients diagnosed by FilmArray were more likely to receive targeted rather than empirical therapy, compared to those diagnosed by culture (P = .0148). Positive Shiga-like toxin-producing E. coli results were reported 47 hours faster with FilmArray and facilitated discontinuation of empirical antimicrobials. Patients diagnosed exclusively by FilmArray had clinical characteristics similar to those identified by culture. Conclusions FilmArray markedly improved clinical sensitivity in patients with acute diarrhea, identified cases with clinical acuity comparable to those identified by culture, and enabled clinicians to make more timely and targeted therapeutic decisions.