Excessive use of chemical fertilizers has deteriorated the conditions of agricultural soils, generating environmental impact has led to the search for alternatives, such as use of biofertilizers using plant growth promoting microorganisms (PGPR), as actinobacteria. Existing studies have shown biomass production difficulties in the substrate stage-separation between biomass, increasing production costs and therefore the aim of this study was to design a culture medium using chemically defined sources for the production of biomass of three isolates of actinomycetes (Thermobifida sp. MCR24, Streptomyces sp. MCR9 and Streptomyces sp. MCR26) and subsequent optimization means MCR26 strain due to its properties as PGPR. It was carried out the selection of carbon sources from a biochemical profile and subsequent application of statistical experimental design using 20 single chemically defined substrates. To optimize, we performed a central composite design, following a response surface methodology. Combinations which generate the maximum response (30 gL-1 of lactose and 4 gL-1 yeast extract, 10 gL-1 glucose, 4 g L-1 yeast extract and 4 g L-1 ammonium sulfate) were validated experimentally obtained with lactose and glucose 3.63 and 2.74 gL-1 gL-1 of biomass respectively. Formulation was achieved two culture media using chemically defined sources of low cost, for the production of biomass from Streptomyces sp. MCR26 PGPR potential.