Using a Taguchi experimental design, this research focuses on utilizing indigenous bacteria from the Danube River to enhance the self-healing capabilities and structural integrity of cementitious materials. Bacillus licheniformis and Bacillus muralis were used as individual bacterium or in co-culture, with a concentration of 8 logs CFU, while the humidity variation involved testing wet and wet–dry conditions. Additionally, artificial neural network (ANN) modeling of the compressive strength of cement samples results in improvements in compressive strength, particularly under wet–dry conditions. By inducing targeted bacterial activity, the formation of calcium carbonate precipitates was initiated, which effectively sealed formed cracks, thus restoring and even enhancing the material’s strength. In addition to short-term improvements, this study also evaluates long-term improvements, with compressive strength measured over periods extending to 180 days. The results demonstrate sustained self-healing capabilities and strength improvements under varied environmental conditions, emphasizing the potential for long-term application in real-world infrastructure. This study also explores the role of environmental conditions, such as wet and wet–dry cycles, in optimizing the self-healing process, revealing that cyclic exposure conditions further improve the efficiency of strength recovery. The findings suggest that autochthonous bacterial co-cultures can be a viable solution for enhancing the durability and lifespan of concrete structures. This research provides a foundation for further exploration into bio-based self-healing mechanisms and their practical applications in the concrete industry.