A series of novel thiazole-pleuromutilin derivatives were designed and synthesized, and their antibacterial activities were evaluated. Most of the synthesized derivatives showed good activity against Gram-positive bacteria, among which compound h19 was more prominent and had the strongest antibacterial activity against MRSA. Compound h19 was selected for further evaluation of bacterial time-kill kinetics, and the results demonstrated its highly promising efficacy in inhibiting MRSA growth. Moreover, h19 exhibited a superior post-antibiotic effect (PAE) value and a lower possibility for bacterial resistance development compared to tiamulin. Docking studies demonstrated the strong affinity of h19 for the 50S ribosomal subunit with a binding free energy of -10.6 kcal/mol. The cytotoxic assay indicated that h19 had low cytotoxicity on both HEK293T and HepG2 cells (IC