Sourdough production is a complex fermentation process. Natural sourdough fermentation without standardization causes great variability in microbial communities and derived products. Starter cultures have emerged as alternatives to natural fermentation processes, which could improve bakery quality and produce bioactive compounds. This study aimed to evaluate the impacts of freeze-drying on the production and viability of sourdoughs with Lactiplantibacillus pentosus 129 (Lp) and Limosilactobacillus fermentum 139 (Lf), as well as their effects on the quality of long-fermentation bread. These strains were selected based on their better performance considering acidification and exopolysaccharide production capacity. Sourdough with Lp and Lf were propagated until the 10th day, when physicochemical and microbiological parameters were determined. The produced sourdoughs were freeze-dried, and bread samples were produced. The freeze-drying process resulted in high survival rates and few impacts on the metabolic activity of Lp and Lf until 60 days of storage. Incorporating Lp and Lf improved the microbiological and physicochemical properties of sourdough and long-fermentation breads. Tested freeze-dried sourdoughs led to reduced bread aging (higher specific volume and decreased starch retrogradation) and increased digestibility. The results show the potential of the freeze-dried sourdoughs produced with Lp and Lf as innovative strategies for standardizing production protocols for the bakery industry, especially for producing long-term fermentation bread.