AbstractCracks in cement-based materials are one of the main factors affecting the durability of structure. Recent research in the field of concrete materials showed that self-healing in cement-based systems can be achieved by triggering biogenic calcium carbonate (CaCO3) precipitation. The goal of this study is to establish a comparative evaluation of the use of sepiolite, bentonite, and diatomaceous earth (DE) as an immobilization barrier of Sporosarcina pasteurii (S. pasteurii) cells to trigger self-healing in cement-based systems. For the first time in the literature, this study will provide insight into the use of natural minerals, such as bentonite and sepiolite, as protective carriers for vegetative S. pasteurii cells in cement-based materials and present a comparative evaluation of factors influencing crack healing, such as the microstructure and composition of immobilization barriers. A two-phase self-healing bioadditive was obtained by immobilizing vegetative S. pasteurii cell samples on natural porous minerals with or without the use of required nutrients. Then the samples were cracked by a three-point bending test, and the healing process was screened via stereomicroscope imaging and ultrasonic pulse velocity (UPV) testing after subjecting the cracked samples to 28 days of moist curing. Flexural cracks induced in mortar samples were filled with biogenic precipitate. Relatedly, the water absorption capacity of the samples was decreased in mortar samples containing bacterial cells, the nutrients were added in the curing solution. Fourier transform infrared spectroscopy and scanning electron microscopy analyses showed that calcite was the predominant polymorph of CaCO3 sealant in cracks.