Alkali-activated slag (AAS) materials are one of the most promising sustainable construction composites. These novel materials are highly characterized by their improved mechanical and durability properties. Nevertheless, the high shrinkage rate hinders their full-scale applications. The low Ca/Si ratio, complex hydration process, and fine pore microstructure are the main causes of the reported shrinkage behavior. This study introduces Bacillus subtilis culture for healing the cracking behavior. The enzymatic action leads to precipitating calcium carbonate crystals that fill AAS cracks and pores. Incorporating calcium oxide has been recommended in multiple studies. The main purpose of adding calcium oxide is to enhance the engineering properties of AAS and provide more calcium ions for the biochemical reactions induced by the added bacteria. However, inconsistent findings about the influence of calcium oxide have been reported. This research provides further insights into the effect of calcium oxide (CaO) on the performance of microbial self-healing efficiency in AAS composite. The results highlight that incorporating calcium oxide as 7% of the binder partial replacement has an 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.