Recently, we developed multifunctional bacterial-laden polymeric fibers (BioFibers) as an innovative delivery system to introduce a bio-self-healing capability into quasi-brittle composites. These BioFibers consist of a load-bearing core-fiber, a bio-compatible hydrogel sheath, and an outer protective damage-responsive shell layer, engineered to endow the matrix with three key functionalities: (i) bio-self-healing, (ii) control over crack growth, and (iii) damage-induced self-activation. This study focuses on evaluating BioFibers’ efficacy in filling cracks within a cementitious matrix. BioFiber-reinforced cement paste samples (BioFRC) were prepared, cracked under controlled flexural conditions, exposed to wet/dry cycles with bio-agent solutions, and monitored for the development of crack-filling efficiency over time. BioFRC specimens were prepared with a BioFiber reinforced zone. Controlled loading was applied to the specimens to induce cracks with the widths of 100–150 μm. Following crack initiation, samples underwent wet/dry cycles: 1 h submerged in a bio-agent solution (urea, yeast extract, and calcium acetate at 20 g/L each), followed by 23 h of dry conditions at 23 ± 1 ℃ for 28 days. Crack-healing efficiency was assessed at 0, 14, and 28-day intervals post-exposure. Moreover, self-healing end-product precipitations were collected from sacrificial samples for material characterization, including thermogravimetric analysis and scanning electron microscopy. Results indicated that precipitations from activated BioFibers achieved a crack-filling ratio of 93.7 ± 3.3% for cracks with an average width of 129 μm after 28 days of exposure. Material characterization tests revealed the formation of calcium carbonate crystals, including combinations of calcite and vaterite, in the healed samples.