In the study, we assessed how acute changes in water temperature and dissolved ammonia concentration can affect the swimming behavior pattern of rainbow trout (Oncorhynchus mykiss). The behavior was analyzed in three different stages: 1) increase stage, (where temperature and ammonia concentration increase during this step, respectively); 2) unchanged stage, (where levels of both factors do not change during this stage); 3) reduction stage, (where ammonia concentration and temperature during this stage are reduced), respectively. The results showed that both factors significantly changed the swimming pattern of the rainbow trout. There were significant differences in swimming parameters (distance from the center, swimming speed, total movement and the average of angular changes of movement) of treated fish in the comparison between treatments, and with the control group. The changes in the swimming pattern of fish in response to physicochemical parameters of water were confirmed to be a good tool in ecotoxicological studies.