Currently, the production of sustainable concrete with high strength, durability, and fewer environmental problems has become a priority of concrete industries worldwide. Based on this fact, the effective microorganism (EM) solution was included in the concrete mixtures to modify the engineering properties. Concrete specimens prepared with 50% fly ash (FA) as an ordinary Portland cement (OPC) replacement were considered as the control sample. The influence of EM solution inclusion (at various contents of 0, 5, 10, 15, 20, and 25% weight) in the cement matrix as water replacement was examined to determine the optimum ratio that can enhance the early and late strength of the proposed bio-concrete. The compressive strength, porosity, carbonation depth, resistance to sulphuric acid attack, and the environmental benefits of the prepared bio-concrete were evaluated. The results showed that the mechanical properties and durability performance of the bio-concrete were improved due to the addition of EM and FA. Furthermore, the inclusion of 10% EM could increase the compressive strength of the bio-concrete at 3 (early) and 28 days by 42.5% and 14.6%, respectively. The durability performance revealed a similar trend wherein the addition of 50% FA and 10% EM into the bio-concrete could improve its resistance against acid attack by 35.1% compared to the control specimen. The concrete mix designed with 10% EM was discerned to be optimum, with approximately 49.3% lower carbon dioxide emission compared to traditional cement.