With the increasing incidence of antibiotic resistance, there is an urgent need to develop new antibiotics with excellent activity against drug-resistant bacteria. Three novel series of tylosin semisynthetic derivatives were designed, synthesized and evaluated for their antibacterial activities against various Gram-positive and Gram-negative bacteria. Among these derivatives, compound C-2 demonstrated potent antibacterial activity against both gram-positive and gram negative bacteria, and non mutagenic. More importantly, compound C-2 displayed high antimicrobial potency against Gram-positive bacteria in a murine model, and was found to be more efficient than tildipirosin. Thus, compound C-2 had great potential as a promising lead compound for the treatment of bacterial infection.