For the creation of healable cement concrete matrix, microbial self-healing solutions are significantly more creative and potentially successful. The current study investigates whether gram-positive “Bacillus subtilis” (B. subtilis) microorganisms can effectively repair structural and non-structural cracks caused at the nano- and microscale. By creating an effective immobilization strategy in a coherent manner, the primary challenge regarding the viability of such microbes in a concrete mixture atmosphere has been successfully fulfilled. The iron oxide nanoparticles were synthesized. The examined immobilizing medium was the iron oxide nanoparticles, confirmed using different techniques (XRD, SEM, EDX, TGA, and FTIR). By measuring the average compressive strength of the samples (ASTM C109) and evaluating healing, the impact of triggered B. subtilis bacteria immobilized on iron oxide nanoparticles was examined. The compressive strength recovery of cracked samples following a therapeutic interval of 28 days served as a mechanical indicator of the healing process. In order to accurately correlate the recovery performance as a measure of crack healing duration, the pre-cracking load was set at 80% of the ultimate compressive stress, or “f c,” and the period of crack healing was maintained at 28 days. According to the findings, B. subtilis bacteria greatly enhanced the compressive strength and speed up the healing process in cracked cement concrete mixture. The iron oxide nanoparticles were proven to be the best immobilizer for keeping B. subtilis germs alive until the formation of fractures. The bacterial activity-driven calcite deposition in the generated nano-/micro-cracks was supported by micrographic and chemical investigations (XRD, FTIR, SEM, and EDX).