Abstract:[Background] Functional mapping is a genome-wide mapping method that uses statistics to analyze the development of dynamic and complex traits in organisms, with the aim of locating quantitative trait loci (QTL) during the development of traits. The application of functional mapping to microbiological studies can unravel complex interactions.[Objective] To locate significant QTLs in the growth and development of two microorganisms by functional mapping, and to identify the key genes affecting the phenotypic growth of the microorganisms functional annotation. [Methods] One hundred strains of Escherichia coli and 100 strains of Staphylococcus aureus were cultured separately or paired together for co-culture. The phenotypic data and SNP data obtained from each strain were used for correlation analysis to locate the significant QTL of the same species under different culture conditions. [Results] The functional mapping identified 217 QTL in E. coli and 152 QTL in S. aureus. The functional clustering and gene annotation analysis showed that scdA, sdrC, sdrD, and ftsA in S. aureus and phr, nagC, eptA, ppsA, priA, and flim in E. coli might play a role in the microbial growth. [Conclusion] We identified the key genes involved in the competitive interactions between E. coli and S. aureus by employing functional mapping. Our results not only provide fundamental data for the application of functional mapping in microbial research but also offer a new idea for deciphering the complex mechanisms of microbial interactions.