The aim of the study was evaluation of the biological activity of 1-(1-arylimidazolidine-2-ylidene)-3-(4-chlorobenzyl)urea derivatives (I – IV) against the Coxsackievirus B3 (CVB3). The cytotoxic activity was tested in HEK293 cell culture. Cells were incubated for 72 hours at 37° C in a 5% CO2 atmosphere. Cytotoxicity of substances was measured by the MTT test. After determining the highest non-toxic concentration of substances their activity against the Coxsackievirus B3 from the Picornaviridae family was estimated. In order to check the antiviral activity of the compounds, a virus suspension was added to the cell culture and incubated for 1 hour at 37 °C. The virus was then removed from the culture and the tested compounds were added. The cell culture was incubated at 37° C in a 5% CO2 atmosphere until the cytophatic effect in the virus control was achieved, and then the virus was titrated. The virus titre was calculated by the Reed-Muench method. EC50 values of compounds I – IV were contained within the range of 211.4 – 359.7 μg/ml. The research demonstrated that only compound II slightly influenced the CVB3 replication by reducing the virus replication level by 0.77 log, which resulted in reducing the titre by 12.8%.