Microbially induced calcium carbonate precipitation (MICP) presents a sustainable, environmentally friendly solution for repairing cracks in cement-based materials, such as mortar and concrete. This self-healing approach mechanism enables the matrix to autonomously close its own cracks over time. In this study, specimens (50 mm in diameter and 25 mm in height) were exposed to submersion and a wet–dry cycle environment. The solution considered a nutrient-rich suspension with calcium lactate, urea, calcium nitrate, and Bacillus subtilis or Sporosarcina pasteurii in a biomineralization approach. The self-healing efficiency was assessed through optical microscopy combined with image processing, focusing on the analysis of the superficial crack closure area. S. and B. subtilis exhibited notable capabilities in effectively healing cracks, respectively, 8 mm² and 5 mm² at 35 days. Healing was particularly effective in samples placed in a submerged environment, especially with a 69 mM concentration of calcium lactate in bacterial suspensions containing B. subtilis, where 87.5% of a 4 mm² crack was closed within 21 days. In contrast, free calcium ions in the solution, resulting from anhydrous cement hydration, proved ineffective for S. pasteurii biomineralization in urea-rich environments. However, the addition of an external calcium source (calcium nitrate) significantly enhanced crack closure, emphasizing the critical role of calcium availability in optimizing MICP for bio-agents in cement-based materials. These findings highlight the potential of MICP to advance sustainable self-healing concrete technologies.