Recently, magnetic iron oxide nanoparticles (IONs) have been used to control and modify the characteristics of concrete and mortar. Concrete is one of the most used materials in the world; however, it is susceptible to cracking. Over recent years, a sustainable biotechnological approach has emerged as an alternative approach to conventional techniques to heal the concrete cracks by the incorporation of bacterial cells and nutrients into the concrete matrix. Once cracking occurs, CaCO3 is induced and the crack is healed. Considering the positive effects of IONs on the concrete properties, the effect of these nanoparticles on bacterial growth and CaCO3 biosynthesis needs to be evaluated for their possible application in bio self-healing concrete. In the present work, IONs were successfully synthesized and characterized using various techniques. The presence of IONs showed a significant effect on both bacterial growth and CaCO3 precipitation. The highest bacterial growth was observed in the presence of 150 mu g/mL IONs. The highest concentration of induced CaCO3 (34.54 g/L) was achieved when the bacterial cells were immobilized with 300 mu g/mL of IONs. This study provides new data and supports the possibility of using IONs as a new tool in designing the next generation of bio self-healing concrete.