In recent years, increasing attention has been paid in veterinary medicine to find novel natural resources to reduce the use of synthetic drugs, avoid side effects, and for better compliance of the animals’ owners. Metronidazole has been used for many years in the treatment of birds’ trichomonosis. Carvacrol is a terpenoid and several biologic activities was attributed to it. The present study developed and characterized a carvacrol nanoemulsion (NanoCAV) and investigated its antitrichomonal activity on Trichomonas gallinae, the causative agent of pigeon trichomonosis, under in vitro condition and compared it with carvacrol (CAV) and the standard antitrichomonal dug, metronidazole (MTZ). Additionally, cytotoxicity of the developed formulation to the fibroblast cell line was evaluated. The NanoCAV mean size and surface charge were 80.5 nm and -31.2 mv, respectively. No significant cytotoxicity was observed for the NanoCAV. Incorporation efficiency of NanoCAV was measured as 75%. Results of antitrichomonal activity assay showed 12 h fifty percent lethal concentrations of 0.39 and 0.27 μg/ml for CAV and NanoCAV, respectively. The NanoCAV based on in vitro activity and low cytotoxicity, can be further studied for its efficacy and safety profile in the pigeons.