<p>Concrete is the backbone of modern infrastructure owing to its strength, availability, and adaptability. Despite these advantages, its susceptibility to cracking remains a major limitation, as cracks enable the ingress of moisture, carbon dioxide, and harmful chemicals, accelerating deterioration. Traditional repair techniques are often expensive, time-consuming, and offer limited long-term serviceability. To address these limitations, a novel technique for bacteria-based self-healing has been developed. Adding bacteria with calcium sources results in calcite precipitation in concrete, which seals the cracks with calcite. The inclusion of fly ash in bacterial concrete (BC) improves both its environmental sustainability and economic viability. This paper discusses the outcomes of an experimental study on Bacterial (<i>Bacillus subtilis</i>) concrete mixes of bacteria concentration of 10⁵ cells/ml in which cement was replaced with four percentages (0,10,15,20) with fly ash (FA) by weight. Tests conducted include compressive strength (CS), flexural strength (FS), electrical resistivity, carbonation, water permeability, and water absorption (WA) for cured samples at 7, 28, and 56 days. Scanning electron microscopy (SEM), Energy dispersive X-Ray (EDX) Analysis, and X-Ray diffraction (XRD) examination were conducted to verify calcite formation in pores, which made the concrete denser. A maximum enhancement in strength among all the concretes was observed at 15% replaced cement. This study reveals that the BC with fly ash achieves higher strength due to calcite precipitation and the pozzolanic activity of fly ash. Reduction in water permeability and carbonation depth, WA makes concrete more durable. The self-healing rate is analysed by measuring crack width using DIGIMIZER software and calculating the unrecovered strength of healed samples of all the mixes. Unrecovered strength is found to be higher in normal concrete (NC) compared to BC, indicating a high rate of healing after adding bacteria with fly ash.</p>