Diagnostic stool multiplex polymerase chain reaction (PCR) testing has attracted considerable interest, because of its high sensitivity, short turnaround time, and ability to detect multiple organisms simultaneously. This study investigates the clinical usefulness of a stool multiplex bacterial PCR in patients with acute diarrhea. We retrospectively evaluated the stool multiplex bacterial PCR results, clinical parameters, and clinical courses of patients hospitalized because of acute diarrhea between August 2014 and November 2016. A total of 725 patients (male, 372; mean age, 30.9 ± 29.3 years) underwent stool multiplex bacterial PCR. A total of 243 pathogens were detected in 226 patients. The detection rate of multiplex PCR testing was higher than that of stool culture (32.7% vs. 3.3%, p < 0.01). Severe symptoms of acute diarrhea (bloody diarrhea, frequent diarrhea) and prescribed empirical antibiotics were significantly more common in the positive multiplex PCR group (p = 0.02, p < 0.01, p < 0.01, respectively). However, mean durations of hospital stay were similar in the 2 groups according to the multiplex PCR results (p = 0.32). In addition, Campylobacter spp., which was the most commonly detected pathogen (97/243, 39.9%), was significantly associated with frequent diarrhea and prescribed empirical antibiotics (p < 0.01), but not with duration of hospital stay (p = 0.09). We concluded that stool multiplex bacterial PCR might be a useful tool for identifying bacterial etiology in patients with acute diarrhea, especially in those with Campylobacter spp. infection.