The North Crimean Canal, which supplies water to the territory of the Crimea, was closed in April 2014. After an eight-year break, in 2022, the Dnieper River water was again channelled along the riverbed of the Canal. After destruction of the Kakhovka hydroelectric power station (June 2023), the water supply stopped. To assess the current state of the canal ecosystem, microbiological studies of its aquatic environment are required. The aim of this study is to assess the current state of the waters in the North Crimean Canal and the adjacent Black Sea waters based on microbiological indicators. Between April 2022 and June 2024, water and sediment samples were collected at 13 stations. The abundance of heterotrophic, hydrocarbon-oxidising, lipolytic and phenol-oxidising bacteria in the water samples was determined using the limiting dilution method on selective media. Temperature, redox potential and dissolved oxygen content were measured. The abundance of heterotrophic bacteria in the canal bed ranged from 4.5·10² to 2.5·10⁷ cells·mL−1. In 85% of the samples, this corresponded to the mesosaprobic zone (10³–10⁵ cells·mL−1). Hydrocarbon-oxidising, lipolytic and phenol-oxidising bacteria were found throughout the study area. The proportion of hydrocarbon-oxidising bacteria within the heterotrophic bacterial community did not exceed 2%, indicating the absence of chronic oil pollution. A positive correlation was found between hydrocarbon-oxidising and lipolytic bacteria (R = 0.73; n = 19; α < 0.05) and between heterotrophic and phenol-oxidising bacteria (R = 0.66; n = 16; α < 0.05). No significant correlation was found between the abundance of the observed microorganisms and parameters such as water temperature, redox potential and pH. The presence of lipolytic, hydrocarbon-oxidising and phenol-oxidising bacteria indicates that the water body under study is undergoing self-purification to remove contamination by the relevant pollutants. Further observations are required for long-term monitoring.