Concrete crack repair materials and technologies have always been the focus of the industry, among which microbial-based concrete crack repair materials and technologies have been widely studied by researchers because they belong to living intelligent materials and technologies. However, the quantitative relationship between the microbial concentration and the amount of substrate used in microbial self-healing concrete was unclear and the depth of repair was shallow, greatly limiting its widespread use. To address these issues, in this work,the mineralisation yield of Bacillus pasteurii was investigated at different substrate concentrations and different bacterial concentrations to clarify the quantitative relationship between bacterial concentration and substrate (Fig. 1), and to investigate the effect of self-healing cracks at different concentrations of bacteria and crack widths. Saccharomyces cerevisiae was then combined with Bacillus pasteurii to form a binary microbial self-healing material to verify the synergistic self-healing effect of the two bacteria on cracks. To further improve the self-healing effect, the binary microbial self-healing material was optimised. Firstly, the two bacteria were domesticated separately for alkaline resistance, and then the best way and ratio of the two bacteria were studied, The results showed that the alkali resistance of Bacillus pasteurii was up to 13, while the highest alkali resistance of Saccharomyces cerevisiae was 11.5, and that the best mixing method was direct mixing before incubation, with mixing ratios of 8:2 and 6:4. The binary microbial self-healing concrete was prepared using the optimised binary microbial self-healing material to investigate the effects of crack width (Fig. 2) and ambient temperature on the repair effect. This study shows that there is great potential for the application of multi-microbial self-healing materials on self-healing concrete, with a view to promoting the practical engineering application of microbial-based concrete crack self-healing materials and technologies.