Cryptosporidium serpentis is a common parasitic disease in captive snakes that is associated with substantial morbidity and mortality. To minimize the impact of this parasite, it is important to identify effective treatment methods. The purpose of this study was to evaluate a new drug regimen for treating C. serpentis in eastern indigo snakes (Drymarchon couperi). Twenty-four eastern indigo snakes naturally infected with C. serpentis were randomly divided into two groups. The first group received 20 mg/kg nitazoxanide, 10 mg/kg azithromycin, and 5 mg/kg rifabutin twice weekly in a food item for 6 wk, whereas the second group received no treatment in the food items. Cloacal swabs were collected every 2 months for 6 months to measure C. serpentis shedding by probe hybridization quantitative polymerase chain reaction (qPCR). The eastern indigo snakes that were qPCR negative after 6 months were immunosuppressed with a single dose of 4 mg/kg dexamethasone sodium phosphate SC. These eastern indigo snakes were then screened by qPCR for an additional 6 months as described previously. Eastern indigo snakes that were qPCR negative after 1 yr of serial sampling were re-evaluated for C. serpentis via gastric biopsy for histological and qPCR analyses. Only 2 (16.7%; 95% confidence interval [CI]: 0.1–37.8) of 12 eastern indigo snakes from each group were qPCR negative before immunosuppression. The eastern indigo snakes in the treatment group did have a decrease in the amount of C. serpentis DNA shedding after treatment (P = 0.025), whereas the control eastern indigo snakes did not (P = 0.232). Only 1 (8.3%; 95% CI: 0.1–23.9) of 12 eastern indigo snakes in each group was negative 6 months after immunosuppression. These findings suggest that 20 mg/kg nitazoxanide, 10 mg/kg azithromycin, and 5 mg/kg rifabutin twice weekly for 6 wk in a food item is ineffective in eliminating C. serpentis in naturally infected eastern indigo snakes.