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Microbially induced calcium carbonate precipitation (MICP) has recently become an intelligent and environmentally friendly method for the repair of cracks in concrete. In order to improve the ability of microbial materials to repair concrete cracks, we applied random mutagenesis and optimization of mineralization conditions to improve the quantity and crystal form of microbially precipitated calcium carbonate. Sporosarcina pasteurii ATCC 11859 was used as the starting strain to obtain the mutant with high urease activity by atmospheric and room temperature plasma (ARTP) mutagenesis. Then, the optimal biomineralization conditions and precipitation crystal form was studied using Plackett-Burman experimental design and response surface methodology (RSM). Biomineralization with 0.73 mol/L calcium chloride, 45 g/L urea, reaction temperature of 45°C, and reaction time of 22 h, significantly increased the amount of precipitated calcium carbonate, which was deposited in the form of calcite crystals. Finally, the repair of concrete using the optimized biomineralization process was evaluated. A comparison of water absorption and adhesion of concrete specimens before and after repairs showed that concrete cracks and surface defects could be efficiently repaired. This study provides a new method for the engineering of biocementing material for concrete repair.