An 8‐year‐old female spayed Labrador retriever was presented for the evaluation of severe weight loss 10 weeks after starting an immunomodulatory treatment, including prednisolone and cyclosporine, for meningoencephalitis of unknown origin. Plasma biochemistry analysis showed mild to moderate increases in liver enzyme activities and a moderate decrease in urea concentration. Abdominal ultrasound revealed mild hepatomegaly and a large gall bladder with unremarkable wall and content. Cholecystocentesis was performed and bile was examined both cytologically and by molecular methods, which revealed the presence of Enterocytozoon bieneusi. Treatment was initiated with albendazole but was discontinued due to the development of severe neutropenia. The medical management was subsequently changed to fenbendazole and the dog made a complete recovery. This report describes the first case of clinical manifestation and successful treatment of biliary E. bieneusi infection in a dog.