Prumycin is a carbohydrate antibiotic. It was isolated from a Streptomyces sp. at Kagawa Prefecture, Japan. It has antifungal, antitumor, and antimalarial activities. The inhibitory properties of prumycin were evaluated in vitro cultures of Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi; furthermore, the in vitro drug combination with clofazimine was assessed for Babesia bovis and Babesia caballi. The IC₅₀ values of prumycin were 22.3, 0.96, 1.89, and 21.17 μM for B. bovis, B. bigemina, B. caballi, and T. equi. The combination of prumycin and clofazimine had a synergetic action on Babesia parasites that improved the potency and decrease the possible toxic side effect in B. caballi and B. bovis cultures. Therefore, prumycin might be of value in blended therapy of babesiosis and theileriosis and further studies are required to evaluate its in vivo effects.