Millions of people die every year due to diarrheal related diseases, with infants and the elderly making up the majority of these deaths. Deaths are caused by excessive intestinal fluid and electrolyte secretion and are especially common in impoverished developing countries. Antibiotics have been classically used as a method to treat diarrhea-related pathologies by modulating the gut microbiome. We recently reported that penicillin may protect against disease-induced excessive fluid and electrolyte secretion via a genetics-independent, microbiome-independent mechanism in individual colonic crypt cells. In this study we investigated whether microbial-independent protective effects of penicillin against fluid secretion can be observed in the rat small intestine at the whole-tissue level. Here we report that penicillin has a significant dose-dependent protective effect against fluid secretion in induced models of diarrhea in the microbiome deficient rat small intestine. Penicillin can rapidly bring fluid secretion down to levels comparable to healthy controls. Our results suggest, for the first time, an alternative function for penicillin G as a cost-effective and fast-acting treatment against diarrheal symptoms without dependence on modulating the behavior of the existing gut microbiome.