| Summary: | This study aimed to evaluate the antibacterial, leishmanicidal, and cytotoxic potential of metabolites produced by bacteria isolated from rhizosphere soil samples. The bacterium was identified by genome sequencing as <i>Streptomyces kronopolitis</i>. A preliminary screening was carried out for the antimicrobial activity of <i>S. kronopolitis</i>, demonstrating activity against <i>Staphylococcus aureus</i> ATCC 6538, <i>Corynebacterium diphtheriae</i> ATCC 27010, <i>C. diphtheriae</i> ATCC 27012, and <i>Mycobacterium abscessus</i>, with inhibition halos of sizes 25, 36, 29, and 33 mm, respectively. To obtain secondary metabolites, the bacteria were subjected to submerged fermentation, and the metabolites were extracted using the liquid–liquid method with ethyl acetate. There was a similar MIC for <i>M. abscessus</i> and the two strains of <i>C. diphtherium</i>, reaching a concentration of 12.5 µg/mL, while that of <i>S. aureus</i> was 0.048 µg/mL. Assays for leishmanicidal activity and cytotoxicity against HEp-2 cells and red blood cells were performed. The metabolite showed an IC<sub>50</sub> of 9.0 ± 0.9 µg/mL and CC<sub>50</sub> of 221.2 ± 7.0 µg/mL. This metabolite does not have hemolytic activity and is more selective for parasites than for mammalian cells, with a selectivity index of 24.6. Thus, the studied metabolite may be a strong candidate for the development of less toxic drugs to treat diseases caused by pathogens.
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