Three Streptomyces strains isolated from Guaviare sediments (Colombia, South America) with cytotoxic activity against prostate cancer (PC3), breast cancer (MDA-MB-231), and lung cancer (A549) line cells were studied. The present investigation reveals the enhancement of the cytotoxic activity evaluating different values of pH, carbon sources (sucrose, glucose, xylose, maltose, and starch), and nitrogen sources (malt extract, yeast extract, meat extract, peptone, and potassium nitrate). Based on the response surface methodology, the isolates Streptomyces aburaviensis (73) had the maximum activity for lung cancer (IC50= 25.00 ± 1.86 ppm) with 4% of yeast extract, 3% of starch, and a pH value of 7. Streptomyces gramineus (386) had the maximum activity against prostate cancer (IC50= 6.14 ± 2.07 ppm) with 5% of malt extract, 3% of glucose, and a pH value of 6. Finally, Streptomyces psammoticus (519) had the maximum activity against breast cancer (IC50= 35.53 ± 2.71 ppm) with 1% of yeast extract, 4% of starch, and a pH 8. The results suggest that the ethyl acetate extracts from isolates Streptomyces aburaviensis (73), Streptomyces gramineus (386), and Streptomyces psammoticus (519) have a potential for use in pharmaceuticals as cytotoxic agents.