Lactobacillus fermentum isolated from sourdough was able to produce riboflavin. Spontaneous roseoflavin-resistant mutants were obtained by exposing the wild strain (named L. fermentum PBCC11) to increasing concentrations of roseoflavin. Fifteen spontaneous roseoflavin-resistant mutants were isolated, and the level of vitamin B₂ was quantified by HPLC. Seven mutant strains produced concentrations of vitamin B₂ higher than 1 mg L⁻¹. Interestingly, three mutants were unable to overproduce riboflavin even though they were able to withstand the selective pressure of roseoflavin. Alignment of the rib leader region of PBCC11 and its derivatives showed only point mutations at two neighboring locations of the RFN element. In particular, the highest riboflavin-producing isolates possess an A to G mutation at position 240, while the lowest riboflavin producer carries a T to A substitution at position 236. No mutations were detected in the derivative strains that did not have an overproducing phenotype. The best riboflavin overproducing strain, named L. fermentum PBCC11.5, and its parental strain were used to fortify bread. The effect of two different periods of fermentation on the riboflavin level was compared. Bread produced using the coinoculum yeast and L. fermentum PBCC11.5 led to an approximately twofold increase of final vitamin B₂ content.