A series of pleuromutilin analogs with a substituted 1,2,4-triazole were designed, synthesized and assessed for their in vitro and in vivo antibacterial activity. Initially, the MIC of the synthesized derivatives against five strains of Staphylococcus aureus (MRSA ATCC 43300, S. aureus ATCC 29213, clinical isolation of S. aureus AD3, S. aureus 144 and S. aureus SA17) were tested by the broth dilution method. Compounds 30a, 31b and 32a were the most active antibacterial agents in vitro against MRSA (MIC = 0.0625 μg/mL). The results of the time-kill curves showed that compounds 30a and 32a could reduce the amount of MRSA in vitro quickly (-7.70 log<sub>10</sub> CFU/mL and -7.10 log<sub>10</sub> CFU/mL reduction). In the experiment to further evaluate the in vivo antibacterial activity of compound 30a against MRSA, compound 30a (-1.71 log10 CFU/g) was effective in reducing MRSA load in thigh infected mice. Compound 30a (survival rate was 50%) displayed superior in vivo efficacy to that of tiamulin (survival rate was 30%) in the mouse systemic model. The results of further pharmacokinetic studies on compound 30a showed that the half-life (t<sub>1/2</sub>), clearance rate (Cl) and the area under the plasma concentration time curve (AUC<sub>0→∞</sub>) of compound 30a were 0.37 h, 5.43 L/h/kg and 1.84 μg h/mL, respectively. After affinity measurement by surface plasmon resonance (SPR), compound 30a exhibited high affinity with the 50S ribosome, with K<sub>D</sub> value of 1.95 × 10<sup>-6</sup> M. Furthermore, the results of molecular docking studies revealed that compound 30a was successfully localized inside the binding pocket of 50S ribosomal subunit (ΔG<sub>b</sub> = -9.40 kcal/mol). Meanwhile, most of these compounds had no significant inhibitory effect on RAW 264.7 cells and 16HBE cells at the concentration of 8 μg/mL. The obtained outcomes showed that compound 30a could be utilized as an encouraging perspective in the development of a new therapeutic candidate for bacterial infection.