Abstract Aim To characterize and compare the bread-making practices and microbial diversity of traditional and bakery sourdoughs from Lebanon and determine how specific practices shape microbial diversity. Methods and results Thirty bread-making practices variables from 26 Lebanese bakers were analyzed using Multiple Correspondence Analysis (MCA) and hierarchical clustering, which revealed three distinct bread-making groups. The first group (wheat flour sourdough) was characterized by the use of wheat flour, frequent backsloppings, high hydration, and fermentation temperatures. The second group (wheat flour Epiphany sourdough) included traditional household practices involving non-backslopped fermented sourdough with lower hydration and fermentation temperatures. The third group (maize flour sourdough) was distinguished by the use of maize flour during sourdough making. Metabarcoding (V3V4 and ITS1) of 50 sourdoughs revealed 141 fungal and 98 bacterial species. In parallel, 351 yeast strains were isolated and identified, providing a valuable Lebanese genetic resource. PERMANOVA on weighted_UniFrac distance clustered bacterial and fungal communities into three significantly different groups, associated with bread-making practices. Non-backslopped “wheat flour Epiphany” sourdoughs exhibited a significantly higher Shannon index compared to the two other sourdough groups. Their microbial communities were characterized by the presence of various bacterial and fungal families, including Enterobacteriaceae, Pseudomonadaceae, Erwiniaceae, Lactobacillaceae, and several filamentous fungi. In contrast, regularly backslopped sourdoughs were dominated by Fructilactobacillus sanfranciscensis and Saccharomyces cerevisiae. These differences in microbial community could be statistically linked to several factors, including temperature and hydration, which were higher in regularly backlopped sourdoughs. Conclusion This study provides the first comprehensive analysis of contrasted sourdough bread-making practices and microbial species diversity in Lebanon and highlight how variations in backslopping practices can significantly influence microbial species diversity and composition in traditional sourdoughs.