Due to rapid urbanization, pavement failure has become a concerning issue in road construction, primarily due to the increase in traffic loading and vehicle volume on roads. The aging process is one of the contributors to pavement failures, as it leads to a loss of the asphalt binder’s ability to hold surface particles together, causing microcracks. Hence, self-healing is an emerging approach that is potentially effective in repairing microcracks in asphalt pavements. The process occurs at an early stage to prevent the development of microcracks, thereby enhancing pavement durability. This study focused on using ureolytic bacteria as a potential self-healing agent in mixtures for hot, warm, and cold mix asphalt. Two methods of incorporating 1% ureolytic bacteria (by weight of the total aggregate) into the mixture were conducted: during mixing and after mixing. Marshall and indirect tensile strength tests were performed. The self-healing mechanism was evaluated by comparing and analyzing the mixture’s performance after one day and seven days of mixing. The findings show that when utilizing the ureolytic bacteria during mixing, the CMA mixtures demonstrated a high potential for self-healing, with a 24% increase in performance after seven days, followed by the WMA and HMA mixtures. This indicates that ureolytic bacteria which undergone process of Microbial Induced Carbonate Precipitation (MICP) have significant potential as a self-healing agent in asphalt mixtures, helping to restore and strengthen roads while reducing microcracks. Consequently, these results promote a sustainable and durable infrastructure, reducing the need for road maintenance in future road applications.