【Objective】 To provide experimental data for the research and development of new antibacterials against Mycoplasma gallisepticum.【Method】Eighteen analogues of pleuromutilin possessing thiadiazole moieties at the C14 chains were synthesized. High resolution mass spectrometry was used to verify the structures of the synthesized compounds. Influence of substituted benzene on the antibacterial effects of these pleuromutilin derivatives against M. gallisepticum were investigated by in vitro drug sensitivity tests.【Result】The obtained MIC values revealed that analogues 12, 15 and 17 exhibited potential antibacterial activities against M. gallisepticum S6 strain with MIC of 0.125 0 μg·mL^-1. Analogue 18 and Tiamulin (control) showed the most potent antibacterial activities against S6 strain in vitro with MIC of 0.015 6μg·mL^-1. Analogue 17, which was the product from benzoylation of the amino group of analogue 18, had lower antibacterial activity compared with analogue 18.【Conclusion】 Analogue 18 can be used as a candidate compound for further development of antibacterials against M. gallisepticum.