Sourdough is one of the oldest microbial biotechnology processes employed by man to leaven bread. The addition of water to grounded cereals followed by restricted fermentation is used as a starter culture for production of sourdoughs. Over the past few decades, there has been a reduction in the diversity of bread aromas and associated flavours due to preference of pure cultures. Use of sourdough starter cultures as a natural leavening agent has many advantages that it offers over baker’s yeast including unique aromas, high tolerance to stressful conditions, a positive influence on the nutritional properties, and prolonged shelf-life of the bread. There is limited information describing microorganisms responsible for spontaneous fermentation of traditional sourdough in Botswana despite its common practice. Information on the yeasts harbored by sourdoughs in Botswana is important to meet today’s customer driven market where diversity of farinaceous products is of utmost importance. This study aims to investigate the microflora found in traditional sourdough and develop a starter culture for making sourdough by identifying the microorganisms responsible for sourdough fermentation, subjecting the isolates to different baking associated stresses, and analyzing the final attributes of the bread.