Our study investigated the behavioural responses of medicinal leech and rainbow trout at different ontogenetic levels under the effect of crude oil (CO) and heavy fuel oil (HFO), performed comparative analysis of the sensitivity of animal responses, evaluated the specificity of these responses, and determined “safe“ toxicant concentrations. Comparison of sensitivity of behavioural responses of leech and fish revealed that the most sensitive response to long-term exposure to CO was leech locomotor activity, while the most sensitive parameter to HFO was the coughing rate in juvenile fish. Our study showed that the sensitivity and specificity of behavioural responses of aquatic animals at different phylogenetic and ontogenetic levels can be successfully used to evaluate the toxicity of ambient water polluted with oil hydrocarbons.