A new pleuromutilin derivative with excellent antibacterial activity, 14-O-[(2-amino-1,3,4-thiadiazol-5-yl) thioacetyl] mutilin (ATTM), may serve as a possible lead compound for the development of antibacterial drugs. However, in vivo efficacy and toxicity evaluations of this compound have not been performed. In this study, we evaluated the efficacy of ATTM by measuring the survival of mice after a lethal challenge with methicillin-resistant Staphylococcus aureus (MRSA), and the 50% effective dose (ED50) was 5.74 mg/kg by the intravenous route. In an oral single-dose toxicity study, ATTM was orally administered to mice at different doses and the 50% lethal dose (LD50) was calculated to be 2304.4 mg/kg by the Bliss method. The results of the subchronic oral toxicity study in rats showed no mortality, exterior signs of toxicity, or differences in the total weight gain or relative organ weights between the treated groups and control group after administration. The hematological and serum biochemical data showed no differences between the treated and control groups, except for the levels of alkaline phosphatase (ALP), creatinine (CR) and blood glucose (GLU), which were significantly different in the high-dose group. The differences in the histopathological findings between the treated groups and the control group were not considered to be treatment-related. Our results indicated that the no observed adverse effect level (NOAEL) for ATTM was 5 mg/kg in this study.