As an alternative on the search for functional food products, this study evaluated the use of sourdough in the preparation of wheat flour tortillas. The sourdough was elaborated with Lactobacillus sanfranciscensis and the wheat flour tortillas were prepared with different concentrations of mother sponge (5%, 15%, and 25%) and fermentation times (1 and 3 h) at room temperature (25 ± 2 °C). Quality (diameter, height, color, pH, stretchability scores, and Kramer shear cell results) of wheat tortillas was evaluated after 24 h of preparation. The mother sponge concentration and fermentation time affected some quality parameters and acceptability properties (taste, aroma, color, opacity, and rollability). In addition, the sourdough tortillas had higher stretchability values than control tortillas. Since most of the prepared sourdough tortillas had acceptability values similar to those of tortilla controls, the introduction of sourdough is a viable means to incorporate additional nutritional and nutraceutical value into wheat tortillas.