Our previous studies showed that the activity of waterlily seeds may prevent diarrhea, against Enteropathogenic E.coli (EPEC) K.1.1 causing diarrhea. Consumption of waterlily seeds for two weeks can prevent diarrhea caused by EPEC K.1.1, and consumption up to 28 days can prevent prolonged diarrhea. The aims of this study was to test the biological activity of pre-cooked waterlily seeds flour as diarrhea prevention. Waterlily seeds was being processed into pre-cooked flour, and the biological activity was tested using rats (Sprague Dawley male rats, weighing 140 ± 5 g) for 28 days. Rats were divided into 3 groups, two control groups were fed as basal diet namely the intervention and no intervention EPEC, and one group having the same diet containing 18.7 g/100g of pre-cooked waterlily seed flour (in isokalori). Male rats were fed for 2,3 or 4 weeks. After 2 weeks treatment feedings, rats were orally infected with E. coli every day of the week. Cecal contents were collected at the end of each time period.The biological activity of treatment was observed by observing body weight, daily feed intake, total microorganisms, total E. coli and total lactic acid bacteria from the cecum contents and histopathological picture of the small intestine (jejunum) of rats. The results showed that administration of pre-cooked waterlily seeds flour could prevent prolonged diarrhea in mice. Substitution pre-cooked waterlily seeds flour in feeding can prevent the occurrence of necrosis of the intestinal mucosal and protect jejunal epithelial from EPEC, that potentially as diarrhea prevention.