Purpose: To optimize the L-asparaginase activity of Actinobacteria isolated from Guaviare river sediments in Colombia.Methods: Actinobacterial strains were evaluated for their L-asparaginase activity using phenol red plates and Nessler’s assays. Strains with L-asparaginase activity were identified based on 16S ribosomal rRNA sequencing, and a central composite design was used to study nutritional and growth factors that could improve L-asparaginase activity. L-asparaginase protein was detected using western blotting and the cytotoxicity of L-asparaginase preparations was evaluated against MDA-MB231 and L929 cell lines.Results: Kitasatospora atroaurantiaca, Streptomyces griseoluteus, and Streptomyces panaciradicis were cultured in medium with lactose as a carbon source and a combination of asparagine and malt extract as nitrogen sources. These strains showed L-asparaginase activities of 29.4, 114.06, and 34.08 U/mg, respectively, and half-maximal inhibitory concentration (IC50) values of 25.61 ± 2.15, 8.18 ± 1.61, and 165.29 ± 1.06 ppm, respectively, against MDA-MB 231 cells. Western blotting analysis revealed the presence of an L-asparaginase monomer with a molecular weight of 37 kDa.Conclusion: Kitasatospora atroaurantiaca, Streptomyces griseoluteus, and Streptomyces Panaciradicis produce L-asparaginases with low L-glutaminase activity and promising cytotoxic activity and thus may be useful for the management of acute lymphoblastic leukemia.Keywords: L-asparaginase, Streptomyces, Kitasatospora, Optimization, Cytotoxicity