Chinese bayberry fruits are used as a folk medicine to cure diarrhea. However, the active compounds have not yet been reported. We found that bayberry fruit extract showed significant antibacterial activity against Salmonella, Listeria, and Shigella, and the minimal inhibitory concentration (MIC) ranged from 2.07 to 8.28 mg/mL. Positive relationships were found between the antibacterial activity and the total polyphenol (r = 0.88) and flavonoid contents (r = 0.92) of samples using different extraction times. The active compounds showed green or blue fluorescence under UV light using the bioautography method and were purified using a polyamide column. The fraction F1 with the most activity was comprised of flavonoids, which included cyanidin-3-O-glucoside, myricetin deoxyhexoside, quercetin-3-O-glucoside, and quercetin deoxyhexoside, and it also possessed an antidiarrheal activity (p < 0.10) at 80 mg/kg in mice. These findings provide scientific evidence for the antidiarrheal function of bayberry.