Mycoplasma synoviae (M. synoviae) infections have become an increasingly serious concern in China because they cause huge economic losses to the poultry industry. Antibiotic treatment is one of control strategies that can be used to contain clinical outbreaks in M. synoviae-free flocks, especially because the bacteria can be transmitted through eggs. To understand M. synoviae infection status in farms of central China and the antibiotic susceptibility of the circulating strains in vivo and in vitro, 485 samples were collected from five provinces from 2019 to 2021. Fifty-two strains were isolated and identified. Determination of the minimum inhibitory concentration (MIC) of eight antibiotics (tylvalosin, tiamulin, tilmicosin, lincomycin, enrofloxacin, chlortetracycline, doxycycline and tylosin) for isolates showed that tylvalosin, doxycycline and tiamulin were effective against 52 clinical isolates (MIC values ≤ 0.0625–0.25 μg/mL, ≤0.0625–1 μg/mL, and 0.25–2 μg/mL, respectively). Tilmicosin, enrofloxacin and lincomycin had high MIC90 values (>32 μg/mL). An artificial M. synoviae infection model was established in chickens for evaluation of the short-term therapeutic effect of these antibiotics. After 5 days of medication, doxycycline (200 mg/L) showed a superior ability to inhibit M. synoviae compared with other groups, as did tylvalosin (200 mg/L). Furthermore, the therapeutic efficacy of tylvalosin (0.4 μg/mL) on intra-embryo-injected M. synoviae was higher than that of tiamulin at the same dose. A combination of MIC values determined in vitro and therapeutic effects observed in vivo revealed that tylvalosin and doxycycline had the best therapeutic effects. Tylvalosin also showed better inhibitory effects on the vertical transmission of M. synoviae than tiamulin.