In the production and management of beef and dairy cattle, controlling diarrhea is one of the important concerns. Pathogenic agents of the disease, protozoan parasites including Cryptosporidium spp., are difficult to control, making prevention, diagnoses, and treatment of diarrhea. In the present study, we investigated a farm with a history of calf deaths over a period of 10 years in order to determine the cause of disease and to clarify the detailed distribution of the pathogens. In four examined calves that were reared in calf pens, all were positive with Cryptosporidium and/or Giardia, while the other breeding stock and adult cattle were negative. Molecular analyses revealed that the isolates from calves were C. parvum subtype IIaA15G2R1 as a zoonotic and G. intestinalis assemblage E. Other pathogenic bacteria and diarrhea-causing viruses were not detected. After treating the calf pens with boiling water and milk of lime (Ca[OH]2), oocysts of C. parvum and cysts of G. intestinalis were not found and no additional calves died. This is the first report to describe the mixed infection of both parasites in Japan.