In this study, we examined 50 bacterial strains collected from five different rhizospheres of cultivated wheat plants and assessed their phosphatase activity. The samples were cultured for a duration of 3 and 7 days in NBRIP medium, with rock phosphate (RP) being utilized as the source of phosphate. The best isolate exhibiting the most activity was identified as isolate no. 19, the best-exhibiting activity which recorded a value of 24.27 and 35.44 mg L^-1 after 3 and 7 days of incubation, respectively. The strain was molecularly identified as Streptomyces sp. MMA using PCR and sequencing of the 16Sr DNA gene, which was entered under accession number OR770185 in the NCBI database. Sequential mutagenesis was performed using ethidium bromide (EtBr) and acridin orange (AO). Several mutants were generated, and the most efficient phosphate solubilizing mutant, designated as mutant Streptomyces sp. AO-31 exhibited phosphatase activity of 60.12 mg L^-1. Response Surface Methodology (RSM) was applied to Streptomyces sp. mutant AO-31. The highest phosphatase-specific activity of 76.55 mg L^-1 was achieved under optimized culture conditions of temperature 37^oC, pH 7, sucrose of 1.5%, yeast extract of 1.5%, 5 days of incubation, and a rock phosphate (RP) concentration of 7 g/L.  Treatments of biofertilizer of rock phosphate with mutant strain Streptomyces sp. AO-31 showed an increase in dry matter yield of wheat plants, P content in wheat plants, P uptake of wheat plants and available soil phosphorus after harvesting, compared to the addition of rock phosphate with wild-type strain Streptomyces sp. MMA-NRC, and the addition of rock phosphate without the addition of biofertilizers.