<p>Microorganisms play a vital role in tobacco growth and processing, yet their correlation with chemical composition during flue curing is understudied. This study examines the dynamic changes in chemical components and bacterial communities during flue curing of lower, middle, and upper leaves. Several key constituents showed distinct changes across leaf positions and curing stages. Xanthophyll decreased during the yellowing stage, while neutral fragrance substances increased after yellowing, especially in middle leaves. Sucrose and proline showed a predominant increasing trend throughout the entire curing process. Nicotine in lower leaves was lowest, and significantly increased by flue curing. Bacterial community analysis revealed higher alpha diversity in lower leaves, dominated by <i>Proteobacteria</i>, with <i>Pseudomonas</i> and <i>Acinetobacter</i> prevalent in lower and middle leaves, and <i>Aeromonas</i> in upper leaves. The flue-curing process, especially the yellowing stage, significantly altered bacterial community structure. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) analysis revealed stage- and leaf position-dependent variations in predicted microbial metabolic functions, with predominant involvement in amino acid, lipid, and carbohydrate metabolism pathways. Specific bacteria, like <i>Flavobacterium</i>, were significantly associated with multiple chemical components, suggesting their potential roles in chemical changes of tobacco leaves during curing. These insights highlight the complex interplay between chemical composition and bacterial dynamics, offering opportunities to optimize the curing process for enhanced tobacco quality.</p>