This study investigates the impact of calcium oxide (CaO) on the microbial self-healing activity of *Bacillus subtilis* in alkali-activated slag (AAS) composites. AAS materials are promising sustainable construction composites known for their improved mechanical and durability properties, but they suffer from high shrinkage rates due to their fine pore microstructure and complex hydration process. The study aims to enhance the engineering properties of AAS by incorporating CaO, which is expected to provide more calcium ions for biochemical reactions induced by the added bacteria. The research highlights that incorporating 7% CaO as a partial replacement for the binder has a significant impact on the engineering properties of bio-AAS materials. The study recommends correlating the percentage of free calcium ions within the AAS mixture with the microbial activity. The results show that while CaO incorporation negatively affects the mechanical properties of bio-AAS composites, it enhances the healing of cracks and improves durability properties such as water absorption, porosity, chloride resistance, and surface electrical resistivity. Microstructural analysis using SEM, EDX, and XRD confirms the formation of calcium carbonate crystals, which fill the cracks and pores, improving the healing efficiency. Despite the decrease in mechanical properties, the treated specimens showed a good potential for crack width reduction and pore healing, with *Bacillus subtilis* successfully precipitating calcite crystals within the crack's mouth.This study investigates the impact of calcium oxide (CaO) on the microbial self-healing activity of *Bacillus subtilis* in alkali-activated slag (AAS) composites. AAS materials are promising sustainable construction composites known for their improved mechanical and durability properties, but they suffer from high shrinkage rates due to their fine pore microstructure and complex hydration process. The study aims to enhance the engineering properties of AAS by incorporating CaO, which is expected to provide more calcium ions for biochemical reactions induced by the added bacteria. The research highlights that incorporating 7% CaO as a partial replacement for the binder has a significant impact on the engineering properties of bio-AAS materials. The study recommends correlating the percentage of free calcium ions within the AAS mixture with the microbial activity. The results show that while CaO incorporation negatively affects the mechanical properties of bio-AAS composites, it enhances the healing of cracks and improves durability properties such as water absorption, porosity, chloride resistance, and surface electrical resistivity. Microstructural analysis using SEM, EDX, and XRD confirms the formation of calcium carbonate crystals, which fill the cracks and pores, improving the healing efficiency. Despite the decrease in mechanical properties, the treated specimens showed a good potential for crack width reduction and pore healing, with *Bacillus subtilis* successfully precipitating calcite crystals within the crack's mouth.