<p>Concrete is one of the most widely used materials globally, yet cracking remains a major issue that undermines its durability and lifespan. Early repair of micro-cracks is essential to prevent their growth and reduce maintenance costs. Incorporating microorganisms and microsilica in concrete, known as bio-concrete, offers an eco-friendly approach for crack healing. This study investigates the optimal concentration of microorganisms and microsilica for enhancing self-healing performance. It also evaluates their effects on mechanical strength and water permeability. Various mix designs with different Bacillus subtilis concentrations and microsilica percentages were tested. The experiments measured compressive and flexural strength, permeability, and the composition of internal precipitates. Results showed that microsilica alone reduced the flexural strength of concrete. However, the SBs10 mix (10¹⁰ cells/ml) exhibited the highest flexural strength and the greatest improvement between 7 and 28 days. The incorporation of bacteria together with microsilica enhanced compressive strength, with SBs12 (10¹² cells/ml) achieving the highest values and continuous strength development over time. This mixture also improved tensile strength and significantly decreased water penetration and absorption. Furthermore, during freeze–thaw testing, SBs10 showed the smallest weight loss, indicating superior durability. A strong negative correlation was found between compressive strength and penetration depth, confirming the effectiveness of the bio-concrete system.</p>