Abstract Background Babesia is a tick-borne protozoan blood parasite that can cause hemolytic anemia, thrombocytopenia, lethargy and splenomegaly in giant pandas. Methods We evaluated the efficacy and safety profile of a therapeutic regimen combining atovaquone and zithromycin in the context of babesiosis in giant pandas that have been naturally infected. The examined pandas underwent clinical and laboratory analyses, including hematology, biochemistry and thyroid hormone profiles. Upon diagnosis, the giant pandas were administered a compounded treatment consisting of atovaquone oral suspension (15 mg/kg, PO, q8 h), azithromycin tablets (10 mg/kg, PO, q24 h) and Enteral Nutritional Suspension (TPF) as a fat-rich supplement (0.5 ml/kg, PO, q8 h) for a 10-day period. Results The combination treatment increased the red blood cell count, hemoglobin levels and hematocrit in the pandas within a short period, while also reducing parasite levels below the PCR detection threshold. Conclusions Our study suggested that atovaquone and azithromycin combination therapy is highly effective for emergency treatment of Babesia sp. infection in giant pandas. Graphical Abstract