Two series of pleuromutilin derivatives were designed and synthesized as inhibitors againstStaphylococcus aureus(S. aureus). 6-chloro-4-amino-1-R-1H-pyrazolo[3,4-d]pyrimidine or 4-(6-chloro-1-R-1H-pyrazolo[3,4-d]pyrimidine-4-yl)amino-phenylthiol were connected to pleuromutilin. A diverse array of substituents was introduced at the N-1 position of the pyrazole ring. The in vitro antibacterial activities of these semisynthetic derivatives were evaluated against two standard strains, Methicillin-resistantStaphylococcus aureus(MRSA) ATCC 43300,Staphylococcus aureus(S. aureus), ATCC 29213 and two clinicalS. aureusstrains (144, AD3) using the broth dilution method. Compounds12c,19cand22c(MIC = 0.25 μg/mL) manifested good in vitro antibacterial ability against MRSA which was similar to that of tiamulin (MIC = 0.5 μg/mL). Among them, compound22ckilled MRSA in a time-dependent manner and performed faster bactericidal kinetics than tiamulin in time–kill curves. In addition, compound22cexhibited longer PAE than tiamulin, and showed no significant inhibition on the cell viability of RAW 264.7, Caco-2 and 16-HBE cells at high doses (≤8 μg/mL). The neutropenic murine thigh infection model study revealed that compound22cdisplayed more effective in vivo bactericidal activity than tiamulin in reducing MRSA load. The molecular docking studies indicated that compound22cwas successfully localized inside the binding pocket of 50S ribosomal, and four hydrogen bonds played important roles in the binding of them.