Substrate and rhizosphere microorganisms are key factors affecting seedling growth; however, the effects of seedling substrates and rhizosphere bacteria on the growth of Quercus variabilis are not completely understood. Here, Q. variabilis seedlings were grown in substrates with and without cork flour, as follows: H substrate (charcoal soil/cork flour/perlite, 1:1:2), S substrate (cork flour/perlite, 1:1), and the control (CK) substrate (charcoal soil/perlite, 3:2). High-throughput sequencing and qPCR were used to investigate the effects of these substrates on seedling growth, physiological indices, and rhizosphere bacterial communities. Root and shoot weights of seedlings grown in H and S substrates were significantly higher than those of seedlings grown in CK. Moreover, H was conducive to chlorophyll synthesis in seedling leaves, and the transpiration rate and intercellular CO2 concentration of the leaves of seedlings grown in H were higher than those of seedlings grown in CK. The number of rhizosphere bacterial 16S rRNA copies was significantly greater in the case of seedlings grown in S than for those grown in H and CK. As well, rhizosphere bacterial richness was higher in seedlings grown in H and S than in those grown in CK. Thus, cork-flour-supplemented substrates are beneficial for seedling growth and development, seedling rhizosphere bacterial abundance and diversity, and the abundance of nitrogen and phosphorus metabolism-promoting microbial taxa.