In recent years, bacteria-based self-healing concrete has attracted considerable attention as an innovative, environmentally friendly, and sustainable technology for enhancing the durability of concrete structures. By utilizing microbial-induced calcium carbonate precipitation, this technology offers a safer, greener alternative to conventional repair methods, reducing maintenance requirements and extending the service life of structures. This study aims to evaluate the crack self-healing efficiency of bacteria-based cement mortar using a Concrete Healing Agent–Polylactic Acid (CHA-PLA) additive as a bacterial healing agent under different environmental conditions by examining compressive strength, crack repair capability, and healing performance. Pre-cracked cylindrical specimens (75 mm diameter × 20 mm height) and 50 × 50 × 50 mm mortar cubes were exposed to full water immersion, laboratory curing conditions, natural outdoor exposure, and seawater immersion. This study demonstrates that the CHA-PLA additive can enhance the self-healing efficiency of cement mortar by sealing cracks and voids up to 0.8 mm in width through calcium carbonate precipitation. The compressive strength results at 7 days showed that mortar specimens containing the CHA-PLA additive exhibited lower compressive strength than the control specimens, with reductions ranging from approximately 1% to 15% depending on the curing environment. Among the investigated environmental conditions, full water immersion provide the highest healing efficiency, while seawater immersion and natural outdoor exposure influence the healing performance due to environmental variability. These findings highlight the critical role of moisture in activating bacterial self-healing mechanisms and demonstrate the potential of the CHA-PLA additive as a sustainable self-healing system for cement-based materials.