This study focused on enhancing the quality of gluten-free rice flour bread by incorporating sourdough fermented with two specific bacterial strains, Lactobacillus fermentum and Lactobacillus plantarum. The research examined the impact of the bacterial type and dough yield (200% and 300%) on sourdough characteristics such as pH, lactic acid concentration, and lactic acid bacteria count. Sourdough prepared at three different concentrations (10%, 20%, and 30%) was then used in baking rice-based gluten-free bread. The results indicated that the highest pH in sourdough (4.53 and 4.60) was associated with samples using a L. fermentum starter at 200% and 300% yield. Meanwhile, the sample made with L. plantarum at 200% yield had the highest lactic acid content (620.33 mg/100 g dry basis). Additionally, an increase in sourdough content in the bread formula resulted in lower pH levels, mold and yeast count, and a* and b* color indices, but higher moisture content and L* color index. A rise in dough yield led to increased pH, baking loss, and mold and yeast count. The highest mold and yeast counts (7.95 log cfu/g) were found in control bread after four days of storage, while the lowest counts (0.72 log cfu/g) were observed in bread made with 30% sourdough containing either bacterial strain at a 200% dough yield on the first day. The sensory acceptance peaked and then decreased with sourdough levels up to 20%. The optimal bread, based on sensory evaluation, was made with 20% sourdough using either L. fermentum or L. plantarum at a 200% dough yield, highlighting the benefits of specific sourdough concentrations and bacterial strains in improving gluten-free bread.