A series of pyridinium cation-substituted pleuromutilin analogues were designed, synthesized and evaluated for their antibacterial activities in vitro and in vivo. Most derivatives showed potent antibacterial activities, especially e4 that displayed the highest antibacterial activity against multi-drug resistant bacteria and was subjected to time-kill kinetics, resistance studies, cytotoxicity and molecular docking assays. Molecular docking results, scanning electron microscopy and o-nitrophenyl-β-galactopyranoside tests showed that e4 not only inhibited bacterial protein synthesis but also disrupted bacterial cell walls. Compound e4 showed an ED50 of 5.68 mg/kg against multi-drug resistant Staphylococcus aureus in infected mice model. In in vivo and in vitro toxicity tests, e4 showed low toxic effects with an LD50 of 879 mg/kg to mice. These results suggest that compound e4 may be considered as a new therapeutic candidate for bacterial infections.