The aim of the present study was to assess the antipyretic activity of the macrolide antibiotic, tilmicosin, at dose levels of 20 and 40 mg/kg of body weight, subcutaneously, in Brewer's yeast-induced fever model in mice. Pyrexia was induced by subcutaneous injection of 20 mL/kg of 20% (w/v) Brewer’s yeast suspension into the animal’s scruff region. Eighteen hours later, feverish animals were treated with either tilmicosin or acetylsalicylic acid (200 mg/kg injectable solution, subcutaneously) or vehicle; and rectal temperatures were evaluated at 1, 2, 3, 4 and 5 h post-treatment using digital thermometers. Tilmicosin showed dose-dependent significant decrease in the elevated body temperature of mice that remained sustained throughout the tested time points from 1 to 5 h in the used model. Both small and large dose levels showed a significant inhibition of elevated body temperature when compared with the corresponding febrile controls (37.65 ± 0.04 vs. 38.41 ± 0.08°C and 37.44 ± 0.04 vs. 38.44 ± 0.04, after 1 h; 37.19 ± 0.04 vs. 38.41 ± 0.08°C and 36.80 ± 0.03 vs. 38.44 ± 0.04°C after 5 h, respectively). These activities were standardized as 38.0 and 51.59% and 47.9 and 66.43% after 5 h, respectively, compared to that of the standard antipyretic and acetylsalicylic acid (200 mg/kg of body weight, subcutaneously). These results may indicate that tilmicosin, in addition to its well established antibacterial activity, possesses significant antipyretic activity that may be beneficial in symptomatic relief when it is used in therapy of infectious disease conditions and inflammatory disorders.