Abstract: Whey, the natural product resulting from coagulation of milk is reported to have diverse pharmaceutical credentials. In the present investigation the anti-diarrhoeal activity of the whey powder was investigated. The Whey powder which was prepared using rennet powder and lactic acid, was studied against Magnesium sulphate-induced Diarrhea in Swiss Albino mice. Castor oil-induced enteropooling studies and in vitro biofilm-forming potentials of the whey powder were also carried out, as this is believed to contribute to the anti-diarrhoeal activities of the preparation. Anti-diarrhoeal activity was more pronounced in mice which received 250 mg kg b.wt. of whey powder when compared to those which received 500 mg kg-1 b.wt. The percentage inhibition of total number of feces in the 250 mg kg-1 b.wt. drug-treated group was 56.14%,whereas the animals which received 500 mg kg-1 b.wt. of whey powder showed 37.18% inhibition. The loperamide treated animal group showed 63.81% inhibition. In castor oil induced enteropooling, the percentage inhibition of intestinal content in the 250 mg kg-1 b.wt. drug-treated group was 61.42% against atropine-treated animal group that showed 26.24% inhibition. The whey powder also exhibited strong biofilm forming capacity with increase in concentration. The anti-diarrhoeal activity of whey preparation established herein is believed to be owing to certain active principles present in it or due to biofilm-forming capacity, which inhibits the attachment of mediators of diarrhoea to mucosal walls of the GI tract or due to interaction of diarrhoea inducing chemicals with whey peptides, which needs further investigation.