Mycoplasma gallisepticum is the major mycoplasmal pathogen in poultry. The present study was undertaken to evaluate the efficacy of six commonly used antimicrobials, including difloxacin, enrofloxacin, ciprofloxacin, doxycycline, gentamicin, and valnemulin, against Mycoplasma gallisepticum, using a Monte Carlo simulation based on the pharmacokinetics-pharmacodynamic (PKPD) theory. The ratio of area under the free concentration vs. time curve for 24 h over MIC (AUC24h/ MIC) served as the PK-PD indices for fluoroquinolones, doxycycline and valnemulin, with the target levels at more than 70 h, 125 h, and 125 h, respectively. The maximum concentration of free drug over MIC (Cmax/MIC) served as the PK-PD index for gentamicin, with the target level at more than 12. In the analysis of drug sensitivity, the MIC50/90 values were as follows: difloxacin, 1/2 μg/mL; enrofloxacin, 0.5/2 μg/mL; ciprofloxacin, 1/2 μg/mL; doxycycline, 2/4 μg/mL; gentamicin, 2/4 μg/mL; and valnemulin, 0.008/0.03 μg/mL, respectively. The probabilities of achieving the PK-PD indices for difloxacin and enrofloxacin were lower (0-6.01%) than ciprofloxacin (27.34-36.67%) which was similar with gentamicin (31.65-45.82%). The lowest probability (0%) was obtained for doxycycline because of its high plasma protein binding, while the highest probability was obtained for valnemulin (90.34-97.95%). The results of this study showed that treatment with valnemulin may be the optimal choice for Mycoplasma gallisepticum infection.