The aim of the present study was to detect the tilmicosin residues in muscles, kidneys and liver of broiler chickens and to investigate its impact on both cardiac enzymes and some hematological parameters. Seventy broiler chickens were divided into two groups; the first was left as a negative control. The second group was given tilmicosin orally (30 mg/kg BW) once/day for three successive days. Reversed phase-high performance liquid chromatography (RP-HPLC) with UV detector at 287 nm and solid phase extraction were used for detecting drug residues in tissue samples. Results indicated a widespread distribution of tilmicosin in most tested tissues. All tissue samples were considered tilmicosin free at the 9 th day after the last oral dose except liver. Tilmicosin elicited a significant reduction in red blood cells count, hemoglobin concentration, packed cell volume, lymphocytes and eosinophiles. On the other hand, heterophiles count and the mean corpuscular volume were increased significantly. White blood cells, monocytes, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration showed non-significant changes. Levels of all cardiac enzymes (Aspartate aminotransferase, lactate dehydrogenase, creatine kinase-MB and Troponin I) were high.