Sourdough applications in bread production are rising in recent decades continuously due to consumers' desire for natural products containing less chemical preservatives. In sourdough, lactic acid bacteria play a key role in the fermentation process. In this study Lactobacillus plantarum and Lactobacillus sanfranciscensis as single starter and their mixture as a mixed starter were used in the preparation of sourdough. The results showed that the use of single starters increased diacetyl and hydrogen peroxide of sourdough and sourdough fermented with mixed starter had the highest content of diacetyl and hydrogen peroxide. In the end sourdough fermentation period the highest lactic acid bacteria count was associated with sourdough containing L. sanfranciscensis. Effects of different starters were significant on the pH of TTA of sourdough, dough and bread, and also resulted in decreased pH and increased of TTA compared to control bread. The results of the assessment of physicochemical and organoleptic properties of bread showed that sourdough containing mixed starter resulted in increasing of height, specific volume and porosity; however, it reduced the hardness of bread crust and crumb, and retarded the emergence of mold colonies. Sourdough bread containing mixed starter gained the highest score of organoleptic properties. Therefore the use of mixed starters consisting of L. plantarum and L. sanfranciscensis could be considered as a suitable starter culture for the production of sourdough and high quality bread