In this study, we investigated the activity of tiamulin fumerate against three strains of Histomonas meleagridis in vitro under different conditions. Tiamulin reduced histomonal growth of all three strains at concentrations of 20 ppm and higher. Cultures in phosphate-buffered saline-based medium were more susceptible than cultures in traditional Dwyers medium. When the cultures were inoculated with higher numbers of histomonads, the activity of tiamulin was reduced. Bacteria present in the cultures were resistant against tiamulin.