Structural cracks are the major cause of structural failure and significantly impact their strength and durability. Bacterial self-healing concrete as a sustainable structural Smart material offers potential advantages by autonomously repairing cracks, reducing repair costs, and eliminating human intervention. Most self-healing concrete bacteria have optimal growth temperatures around 25–37°C. Their metabolic activity can decline at higher temperatures, potentially affecting their ability to produce calcium carbonate and heal cracks. This study aimed to investigate the effectiveness of heat-resistant bacteria strains in self-healing concrete under high temperatures. The study examined the impact of temperature, bacteria type, and content on self-healing concrete properties at four different temperatures: room temperature, 60°C, 120°C, and 200°C. Several tests were performed to test the effectiveness of the bacteria strains, including compressive strength tests, scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and X-ray diffraction (XRD). This study showed significant improvement in the mechanical strength of the concrete, particularly at 60°C by 91.2% for compressive strength and 95.95% for indirect tensile strength. The SEM and EDS tests also revealed the presence of calcium carbonate, which suggests that the bacteria were actively involved in the self-healing process. However, as the temperature increased to 200°C, there was a noticeable decrease in strength compared to 60°C, but the compressive strength at room temperature remained higher, indicating that heat-resistant strains of bacteria may not be effective at higher temperatures.