Aims: The study is to characterize the local strain of Rhodococcus sp. – 3/4/3, isolated from environmentally polluted wastewater, to study the ability of the strain to synthesize BS. Methodology and results: The natural strain of Rhodococcus sp. - 3/4/3, a biosurfactant (BS) producer, was isolated from the polluted wastewater of Navoiyazot JSC. Based on morphological, cultural, physiological, and biochemical properties, sequencing the gene of the 16S rRNA strain was identified as Rhodococcus ruber - 3/4/3. The study used qualitative and quantitative methods for the biosynthesis of biosurfactants by Rhodococcus sp. – 3/4/3. Conclusion, significance, and impact of study: It has been established that the strain Rhodococcus ruber - 3/4/3 is a BS producer. For BS biosynthesis, the strain was grown on a medium with the simultaneous use of a hydrophilic 2% hexadecane (HD) and a hydrophobic carbon source of 1% glucose, and 0.1% urea was used as a nitrogen source. It was shown that the strain Rhodococcus ruber - 3/4/3 synthesizes cell-bound BS, and BS biosynthesis is associated with the amount of cell biomass. BS was extracted with methyl tert-butyl ether (MTBE), and thin layer chromatography (TLC) revealed only one fraction of glycolipids with Rf=0.64, the yield of partially purified biosurfactant was 4.8 g/l.