Amoebae of the Acanthamoeba castellanii Neff strain, cultured in bacteria-free condition at room temperature, were tested in vitro for their susceptibility to selected chemical compounds. The amoebae, grown during 4 and 10 days following regular subculturing, were exposed for 24 h to three compounds: the newly synthesized imidazole derivatives (1) 2-methyl-3N-(4-nitrobenzyl)- 5-nitro-1H-imidazole designated as AG16; (2) 2-methyl-3N-(3,5-dinitrobenzyl)-5-nitro-1H-imidazole designated as AG17 and to the (3) cationic antiseptic agent chlorhexidine digluconate (CHX). Two different concentrations of the above-mentioned substances were applied. The results showed that all chemicals tested had some amoebicidal effect on 4-day grown population of Acanthamoeba; variations in susceptibility of the amoebae, depending on the kind and concentration of the compounds occurred. It was also observed that several agents changed the relationship between A. castellanii trophozoites and cysts. Chlorhexidine digluconate caused a decrease in percentage of the trophozoites to 88.4% at higher concentration of the compound, 10 ug/ml (in comparison to 97.8% from the control samples), however, significant increase of the cyst percentage, to 11.6% in assays with this concentration of chlorhexidine were revealed (2.2% in the control samples). Tendency toward induction of encystment was also apparent when AG17 was applied. Because activation of the dormant cysts can lead to repeated development of amoebae, very important is cysticidal efficacy of tested agents. Among all compounds examined by us, the newly synthesized imidazole derivative AG16 was the most effective. It was expressed as decrease in average number of amoebae (to 655.6 × 10³/ml at concentration of the agent 8 ug/ml; 682.5 × 10³/ml in assays with CHX at concentration 4 ug/ml; 893 × 10³/ml in the control samples) connected with a clear cysticidal effect (1.4% of cysts at concentration 4 ug/ml of AG16).