Representatives of Streptomyces genus belong to Actinobacteria family and produce a wide range of biologically active substances (BAS) with diverse biological properties, which are use in various fields of agriculture. The new Streptomyces netropsis IMV Ac-5025 strain was isolated by the researchers from Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine which is antagonist against phytopathogenic fungi (representatives of Alternaria, Fusarium, Cladosporium genera) and synthesizes the polyene antibiotic complex (heptaene candidine and tetraene), phytohormones, and others BAS. However, the relationship between the producer's biosynthesis of polyene antibiotics and other metabolites and their effect on the yield of the target bioproduct is not clear over time. The correlation analysis of the relationship between the biosynthesis pathways of different classes of biologically active substances produced by S. netropsis IMV Ac-5025, conducted by authors, showed that β-sitosterol has a significant effect on the accumulation of polyene antibiotics. Therefore, the aim of the work was to analyze the effect of exogenous β-sitosterol on the accumulation by S. netropsis IMV Ac-5025 of polyene antibiotics and other biologically active substances de novo under the conditions of submerged cultivation. The polyene antibiotic is an intracellular metabolite, but the addition of a steroid compound has caused their super synthesis and excretion from the cell. The amount of polyene antibiotics in the culture liquid of S. netropsis IMV Ac-5025 ranged from 3 to 13.38 μg/ml and was the highest under the action of 10 μg/ml of β-sitosterol, which is 4.46-fold more than in the control variant. The accumulation of both fractions: heptaene candidine and tetraene was increased. Under the action of 0.1; 0.25; 2.5; 5; 10 μg/ml of exogenous β-sitosterol in the producer's biomass polyenes were accumulated and ranged from 2315 to 4919 μg/g, whereas in the control their quantity was occurred 1929 μg/g of absolutely dry biomass (ADB). Under the action of10 μg/ml of exogenous substance, the amount of tetraene was increased 2.3-fold and candidine – 3.2-fold, and the amount of polyenes – 2.5-fold compared control variant. Exogenous β-sitosterol had a positive effect on the accumulation of auxins (indole-3-acetic acid, indole-3-acetic acid hydrazide, indole-3-carbinol, indole-3-carboxylic acid, indole-3 butyric acid, indole-3-carboxaldehyde) and abscisic acid (ABA). The amount of auxins in the biomass of producer was ranged from 56.14 to 458.4 μg/g ADB and was the highest under the action of 5 μg/ml of β-sitosterol, which is 8-fold higher than without the addition of the substance. The ABA accumulation was ranged from 3.2 to 8.1 μg/g ADB and was highest under the action 0.25 μg/ml of β-sitosterol, which is 2.5-fold higher than in the control variant. Thus, the addition of β-sitosterol in the synthetic nutrient medium during the submerged cultivation of S. netropsis IMV Ac-5025 increases the biosynthesis of both polyene antibiotics (2.5fold) and phytohormones, including auxins (8-fold) and ABA (2.5-fold) in the producer's biomass, which may indicate the relationship between the pathways of their biosynthesis. This creates the preconditions for metabolic regulation of qualitative and quantitative accumulation by S. netropsis IMV Ac-5025 of biologically active substances (antibiotics, phytohormones, etc.) and obtaining a bioproduct with specified properties for application in agriculture.