Pollution of water resources with pesticides negatively affects aquatic organisms and makes water bodies unsuitable for use by humans. Chemical analysis methods do not provide information about the impact of a detected substance and its individual components on the ecosystem. The article presents an analysis and synthesis of published data on the possibilities and features of the use of aquatic enzymes as biomarkers of pollution of aquatic ecosystems with pesticides. Publications over the past 20 years, indexed in the PubMed, Crossref, Web of Science, Scopus, and RSCI databases devoted to the issues of pesticide pollution of water bodies and the use of enzymes, in particular nonspecific esterases, in biomonitoring were analyzed. The concept of “biomarker” is revealed, groups of biomarkers, the purposes of their use, advantages and disadvantages as a source of information about the state of the ecosystem are listed. Particular attention is paid to study of nonspecific esterases in aquatic organisms (fish, mollusks, crustaceans, amphibians). The main types of substrates used to measure the activity of esterase isoforms and the features of changes in enzymatic activity in response to exposure to pesticides from different chemical groups (organophosphorus compounds, carbamates, pyrethroids) are considered. The factors influencing the activity of nonspecific esterases of aquatic organisms and limiting their use for assessing pollution of aquatic ecosystems are identified.