The growing demand for eco-friendly and carbon-neutral concrete technologies has driven interest in microbial solutions for CO₂ sequestration and self-healing properties. Microbially induced calcium carbonate precipitation (MICP) is a promising biomineralization process in which specific microorganisms hydrolyze urea via urease enzymes, increasing pH and promoting calcium carbonate formation. This precipitate fills pores and cracks in concrete, enhancing durability and enabling self-repair. In this study, microorganisms were isolated from waste concrete, yielding a total of 42 isolates. Biological analyses identified 11 distinct strains, from which those with high urease activity or spore-forming ability for alkaline survival were selected. The selected strains were tested in CaCl₂–Na₂CO₃ media, revealing that one strain exhibited the highest biomineralization efficiency. Genomic analysis identified a complete urease gene cluster (ureA–ureC structural genes and ureD, ureE, ureF, ureG maturation genes), with genetic variations influencing ureolytic activity. Additionally, genes such as nhaC, involved in pH homeostasis, and mgtE, regulating Mg²⁺ for membrane stability, were found to contribute to performance in alkaline concrete environments. These functional and genomic insights position the strain as a strong candidate for microbial concrete enhancement. Future work will focus on improving strain viability within concrete and validating its performance in real structures. This research advances sustainable construction materials by enabling enhanced durability, reduced carbon emissions, and the potential for large-scale CO₂ mitigation in the concrete industry.