The current study examines the effect of novel microorganisms at different concentrations for use in microbial concrete, including two types of gram-positive bacteria (namely, Streptomyces geysiriensis and Staphylococcus aureus (MRSA)) and two different fungal species (namely, Trichoderma reesei ATCC13631 and Aspergillus nidulans MAD144). The results indicate that bio-concrete, at all concentrations, has more strength than normal concrete. The specimens with Trichoderma reesei exhibited a maximum increase in compressive strength and split tensile strength by 45.95% and 23.3%, respectively. Moreover, non-accelerated samples showed a significant decrease in corrosion rate when using all the introduced microorganisms. The study also found that microbial calcite precipitation was present in the pores of the concrete. The study’s findings emphasize that the introduced microorganisms can be used for microbial concrete with improved mechanical properties and crack healing. Trichoderma reesei has shown the greatest effectiveness in improving concrete’s resilience to corrosion, self-healing capacity, and durability.