It is known that microorganisms play an important role in the ecological and biochemical processes which take place on the Earth. Moreover, Actinobacteria are the most important component of the microbial communities responsible in the aquatic ecosystems for the processes of mineralization of organic substances and recycling of nutrients. The study of the microflora in the waters of lakes throughout the Earth showed among the most common types Proteobacteria, Verrucomicrobia, Cyanobacteria and especially Betaproteobacteria (up to 60-70 % of the total number of cells), the number of which is high due to increased concentrations of various pollutants in the water. Recent data on the composition of freshwater bacterial communities have shown that representatives of Actinobacteria dominate against the background of Betaproteobacteria, Acidobacteria, Firmicutes, and Chloroflexi (Eiler, 2005; Lliros, 2014; Benson, 2019; Kurilkina, 2016). The purpose of the research was to study the diversity of Actinobacteria isolated from the biofilm, water column, and the silt bottom sediments of the "La Izvor" lake system in the Chisinau city. For isolation and quantification of Actinobacteria, the method of inoculation on selective agar media was used. For suppressing the growth of other microorganism groups, antibiotics were added. Additionally, compounds that promote the growth of representatives of actinobacteria (soluble starch, oat flour, propionic acid, yeast extract, etc.) were added. This made it possible to identify new strains of Actinobacteria – representatives of 8 genera, most often found in the microbial communities of the soil and aquatic environment of the Republic of Moldova: Actinomadura, Actinoplanes, Frankia, Geodermatophilus, Micromonospora, Nocardia, Rhodococcus, and Streptomyces. It was found that most of the representatives of Actinobacteria were found in the silt bottom sediments and the water column, and least of all in the biofilm. For example, it was not possible to identify representatives of the genus Actinomadura and Rhodococcus in the biofilm. Actinobacteria of the genus Actinoplanes, Frankia, Geodermatophilus, Micromonospora, and Nocardia were identified by 1 strain each in biofilm samples, while the genus Streptomyces was represented by 4 strains, characterized by active growth of substrate and aerial mycelium on the used agar media. Taking into account the ability of Actinobacteria to synthesize substances that are effective means of combating various pathogens (which cause diseases of plants, animals and humans), as well as amino acids, lipids and enzymes; it seems appropriate to deposit the new strains of Actinobacteria isolated from the "La Izvor" lake system in the National Collection of NonPathogenic Microorganisms and to test them as potential producers of biologically active substances of various chemical nature.