The incorporation of microorganisms into cementitious matrices has been explored as a strategy to enhance the mechanical properties and durability of concrete. Under suitable conditions, microorganisms are capable of producing calcium carbonate, which leads to matrix densification and enables the self-healing of the material. This study investigates the effects of incorporating Bacillus sp. bacteria into cement paste as an admixture, aiming at future applications in concrete with bio-aggregates derived from biological waste. A paste composed by mass of 40% cement, 30% fly ash, and 30% metakaolin was used, with a water-to-binder ratio of 0.4. Initially, an analysis was conducted to determine the optimal bacterial content in the matrix through compressive strength tests on cement pastes with varying percentages (0%, 0.001%, 0.01%, 0.1%, and 0.5%) of bacterial dry mass in the mixing water. Additionally, mixtures were prepared with 1% calcium acetate and 1% calcium acetate combined with 1% nutrient broth to assess the bacteria’s survival capacity in the cementitious matrix and their ability to produce calcium carbonate. Rheological tests indicated lower viscosity for pastes with the addition of calcium carbonate and nutrient broth, suggesting improved workability. Compressive strength improved at intermediate bacterial concentrations (0.01% and 0.1%), while higher concentrations led to reduced performance. The presence of calcium acetate combined with nutrient broth improved compressive strength for all bacterial concentrations. The incorporation of organic components for the bacteria improved the studied properties, and an intermediate bacterial concentration proved to be the most suitable for incorporation into the cementitious matrix.