A major concern today is the pollution of freshwater ecosystems caused by human activities. It is of great importance to protect and prevent further degradation. In this paper, the applicability of the predatory water beetle Cybister lateralmarginalis as a model species for biomonitoring of freshwater habitats in Croatia was investigated. The research was carried out in Pannonian Croatia during the spring-summer season of 2023. The species was sampled at different locations along three large Croatian rivers: Sava, Drava and Danube, and protocols were established for measuring the activity of AChE (acetylcholinesterase), CES (carboxylesterase), GST (glutathione -s-transferase) and concentrations of GSH (glutathione) and ROS (reactive oxygen species). The analyzed biomarkers are used as early warning signs of the presence of various pollutants in the environment, which negatively affect the health of living organisms. CES proved to be a more sensitive biomarker than AChE. Analysis of biomarkers of oxidative stress, GST, GSH and ROS showed a significant difference between the investigated areas. An elevated response indicates the presence of various environmental stressors and a changed state of the environment. The researched species of predatory water beetle is a relatively long-lived predator, and due to its wide distribution and abundance, as well as the simplicity of sampling, it is an appropriate model species for monitoring freshwater areas. It is proposed to introduce the evaluation of the response of biomarkers into the regular monitoring of freshwater ecosystems in order to deepen knowledge and obtain a complete picture of the impact of pollutants on the living world.