Sour cassava starch is a product obtained from cassava (Manihot esculenta Crantz) processing to overcome raw material toxicity and low nutritional value (lipids and proteins). It has the potential to succeed in the international market due to its innovative use in gluten-free baked products. However, the requirements of this market are high and a standardized fermentation process is necessary to obtain an innocuous product of homogeneous quality. The aim of this work was to produce sour cassava starch in a pilot-scale fermentation process with Lactobacillus plantarum as a single culture and in co-culture with Pichia scutulata and to determine the fatty acids and volatile compounds profile. The starter cultures contributed to standardization because, regardless of the culture tested, it was possible to obtain sour cassava starches safe for consumption (absence and low counts of pathogens) with satisfactory acidity (between 1and 5 %) and expansion capacity (higher than 1.00). The products obtained with single and mixed cultures exhibited a reduction of saturated fat (3.6 % and 8.1 %, respectively), an increase of the unsaturated fat (2.3 times), and an increase of the oleic, linoleic and linolenic acids proportion. These fatty acids have a positive effect on human health and wellbeing. A greater variety of desirable volatile compounds was a differential of the product obtained with the mixed culture. Therefore, L. plantarum with P. scutulata was the starter culture that exhibited better results in the pilot-scale fermentation process and has the potential for the industrial production of sour cassava starch.