After a single oral treatment, the pharmacokinetic characteristics of tylvalosin alone and in combination with either amprolium or toltrazuril in Mycoplasma gallisepticum-infected broiler chickens were evaluated. 18 chickens were divided into 3 equal groups, group one was given tylvalosin only, group two was given tylvalosin and amprolium and group three was given tylvalosin and toltrazuril. The best model for describing tylvalosin serum levels in our study was found to be a two-compartment open model. The maximum serum concentration of tylvalosin was 1.62±0.03, 1.2±0.02, and 1.41±0.01μg/ml reached at 2.95±0.014, 2.8±0.01, and 2.83±0.02 h in group one, group two, and group three respectively. A significant (P<0.001) increases in V/F and Cl/F 10.19±0.11(mg)/(μg/ml), 1.54±0.04(mg)/(μg/ml)/h and 8.56±0.07(mg)/(μg/ml), 1.31±0.03(mg)/(μg/ml)/h were found in group two and group three, respectively in comparison with group one 7.24±0.06(mg)/(μg/ml), 1.09±0.024 (mg)/(μg/ml)/h. The absorption and elimination half-lives of tylvalosin were 1.69±0.016h, 10.63±0.330h and 1.56±0.01h, 9.63±0.41h and 1.58±0.02 h, 9.64±0.35 h in group one, group two, and group three respectively. The area under the curve was significantly (P<0.001) decreased in group two and group three which were 13.6±0.06 and 15.98±0.14 μg/ml*h respectively, in comparison with group one which was 18.7±0.093 μg/ml*h. It was found that the serum concentration of tylvalosin in group one after twenty-four hours is higher than MIC for the used Mycoplasmagallisepticum  (0.25 μg/ml) unlike the second and third group, so it is recommended to give tylvalosin every twenty-four hours if it is given alone while when it is given with amprolium and toltrazuril it should be given every twelve hours.