<p>This study evaluated the crack-healing potential of cementitious matrices using different biological agents and application methods. The research involved the production of prismatic mortar samples (40 x 40 x 160 mm) were produced with a 1:3 (cement: sand) ratio and a 0.48 w/c ratio, with the addition of 10⁵ bacterial spores mL⁻¹ and 1% nutrients (yeast extract and calcium acetate) concerning the cement mass. Spores from 5 different biological agents (B1, B2, B3, B4, and B5) were applied in three ways: mixing water (AM), spraying (ASP), and mixing water and spraying (AM-ASP). Self-healing was evaluated in non-standard flexural induced cracks over 120 days by stereomicroscope image analysis, analysis of calcium carbonate presence using the thermogravimetric analyses (TGA), and the precipitate’s mineralogical composition evaluation using the X-ray diffraction (XRD) assay. The bacteria studied performed well in the biodeposition of calcium carbonate in the cement matrix for the three types of application tested. The <i>Bacillus subtilis</i> (B1), <i>B. subtilis</i> (B2), and <i>B. cereus</i> (B4) bacteria stood out the most when applied to the mixing water and the combination of mixing water with spraying. For the period evaluated, samples with small cracks (less than 0.43 mm) were more successful in self-healing. Using <i>Bacillus</i> species bacteria as biological self-healing agents in cementitious matrices could be a promising solution for minimizing pathological manifestations resulting from cracks.</p>