Lactic acid bacteria (LAB) were obtained from durum wheat flour samples and screened for roseoflavin-resistant variants to isolate natural riboflavin-overproducing strains. Two riboflavin-overproducing strains of Lactobacillus plantarum isolated as described above were used for the preparation of bread (by means of sourdough fermentation) and pasta (using a prefermentation step) to enhance their vitamin B2 content. Pasta was produced from a monovarietal semolina obtained from the durum wheat cultivar PR22D89 and, for experimental purposes, from a commercial remilled semolina. Several samples were collected during the pasta-making process (dough, extruded, dried, and cooked pasta) and tested for their riboflavin content by a high-performance liquid chromatography method. The applied approaches resulted in a considerable increase of vitamin B2 content (about 2- and 3-fold increases in pasta and bread, respectively), thus representing a convenient and efficient food-grade biotechnological application for the production of vitamin B2-enriched bread and pasta. This methodology may be extended to a wide range of cereal-based foods, feed, and beverages. Additionally, this work exemplifies the production of a functional food by a novel biotechnological exploitation of LAB in pasta-making.