A series of novel acetamine phenyl pleuromutilin derivatives incorporating 2-aminothiophenol moieties into the C14 side chain were synthesized via acylation reactions under mild conditions. The in vitro antibacterial activities of the synthesized derivatives against three Staphylococcus aureus (MRSA ATCC 43300, ATCC 29213 and AD 3) and two Escherichia coli (ATCC 25922 and 9-1) were evaluated by the broth dilution method. Most of the synthesized derivatives displayed potent activities. Compound 27 was found to be the most active antibacterial derivative against MRSA (minimal inhibitory concentration = 0.015 μg/mL) which may lead to a promising antibacterial drug. Furthermore, compound 27 displayed more rapid bactericidal kinetic than tiamulin in in vitro time-kill studies and possessed a longer PAE than tiamulin against MRSA. The PK properties of compound 27 were then measured. The half life (t1/2), clearance rate (Cl) and the area under the plasma concentration-time curve (AUC0→∞) of compound 27 were 6.88 h, 21.64 L/h/kg and 0.48 μg h/mL, respectively. The in vivo antibacterial activities of compound 27 against MRSA were further evaluated using thigh infection model and systemic infection model. Compound 27 possessed superior antibacterial efficacy to tiamulin against MRSA infection in both model.