Abstract: This study addresses the issue of low Li₂O recovery in concentrate, leading to lithium resource waste, in a lepidolite processing plant in Changde, Hunan Province. Taking the middlings stream before regrinding as the research object, we conducted a comparative study between conventional grinding—flotation and microbial pretreatment—flotation processes. Using a closed−circuit flowsheet comprising one roughing, one cleaning, and two scavenging stages with sequential middlings return, the microbial pretreatment−flotation process achieves a concentrate grade of 3.02% Li₂O with a recovery of 79.81%. Compared to the conventional grinding−flotation process, this approach increases Li₂O recovery by 15.21%. Furthermore, the dosages of collector and dispersant are reduced by 25% and 46.15%, respectively. The results demonstrate that the growth and metabolism of silicate bacteria cause solubilization and decomposition of mineral particles. This process increases the degree of liberation of mineral monomers and exposes the 001 solvation surface, facilitating the selective adsorption of amine collectors onto zinnwaldite. The microbial pretreatment process avoids the problems of high energy consumption and severe fine mud generation problems caused by grinding, effectively enhancing the economic efficiency of the comprehensive recovery of lithium resources.