This study investigates the impact of calcium oxide (CaO) on the microbial self-healing efficiency of alkali-activated slag (AAS) composites. AAS is a promising sustainable construction material with improved mechanical and durability properties, but its high shrinkage rate limits its application. The study introduces Bacillus subtilis to enhance the self-healing process by promoting the precipitation of calcium carbonate crystals that fill cracks and pores. The addition of CaO is recommended to improve the mechanical properties of AAS and provide more calcium ions for microbial reactions. However, the effect of CaO on microbial self-healing is inconsistent, with some studies showing positive outcomes and others negative effects. The research found that incorporating 7% CaO as a binder replacement significantly affected the mechanical properties of bio-AAS materials, leading to a 21.6% reduction in 28-day flexural strength. The addition of CaO increased free calcium ions, which promoted the formation of metal hydroxides, disrupting microbial reactions. High silicate concentrations led to rapid silicate gelation, increasing porosity. The RCPT results showed that the presence of mobile ions like Na⁺, OH⁻, and HS⁻ affected chloride resistance, while CaO addition reduced chloride penetration by reacting with these ions. Durability tests revealed that CaO addition increased water absorption and porosity, but the microbial activity partially offset these effects by filling pores. Surface resistivity tests indicated good chloride resistance, consistent with AAS's known high resistance to chloride attack. XRD and SEM analyses confirmed the formation of calcium carbonate and other hydration products, highlighting the role of microbial activity in self-healing. However, the presence of CaO also led to the formation of brittle compounds like portlandite, which could weaken the composite. The study concludes that while CaO enhances microbial self-healing by providing calcium ions, it also has negative effects on mechanical properties. Further research is needed to optimize the concentration of CaO for optimal microbial efficiency in AAS composites. The findings emphasize the importance of balancing CaO addition with microbial activity to achieve effective self-healing in AAS materials.This study investigates the impact of calcium oxide (CaO) on the microbial self-healing efficiency of alkali-activated slag (AAS) composites. AAS is a promising sustainable construction material with improved mechanical and durability properties, but its high shrinkage rate limits its application. The study introduces Bacillus subtilis to enhance the self-healing process by promoting the precipitation of calcium carbonate crystals that fill cracks and pores. The addition of CaO is recommended to improve the mechanical properties of AAS and provide more calcium ions for microbial reactions. However, the effect of CaO on microbial self-healing is inconsistent, with some studies showing positive outcomes and others negative effects. The research found that incorporating 7% CaO as a binder replacement significantly affected the mechanical properties of bio-AAS materials, leading to a 21.6% reduction in 28-day flexural strength. The addition of CaO increased free calcium ions, which promoted the formation of metal hydroxides, disrupting microbial reactions. High silicate concentrations led to rapid silicate gelation, increasing porosity. The RCPT results showed that the presence of mobile ions like Na⁺, OH⁻, and HS⁻ affected chloride resistance, while CaO addition reduced chloride penetration by reacting with these ions. Durability tests revealed that CaO addition increased water absorption and porosity, but the microbial activity partially offset these effects by filling pores. Surface resistivity tests indicated good chloride resistance, consistent with AAS's known high resistance to chloride attack. XRD and SEM analyses confirmed the formation of calcium carbonate and other hydration products, highlighting the role of microbial activity in self-healing. However, the presence of CaO also led to the formation of brittle compounds like portlandite, which could weaken the composite. The study concludes that while CaO enhances microbial self-healing by providing calcium ions, it also has negative effects on mechanical properties. Further research is needed to optimize the concentration of CaO for optimal microbial efficiency in AAS composites. The findings emphasize the importance of balancing CaO addition with microbial activity to achieve effective self-healing in AAS materials.