There is a growing interest in the development of flours from sprouted cereals due to their enhanced nutritional and bioactive potential, but this research field has been scarcely explored. This study aimed to investigate the influence of germination conditions on protein profile, bioactive potential and enzymatic activities related with quality in oat gluten-free flours. Germination temperatures and duration ranging 12–20 °C and 24–216 h, respectively, were studied in hulled and dehulled oat varieties. Germination increased free phenolics, antioxidant capacity, protein hydrolysis and α-amylase activity in oat flours, particularly at high temperatures and longer times. β-glucan content was retained at short germination times (24–96 h) but it was reduced beyond this time period. Lipase activity increased during germination in the hulled variety and declined in the dehulled one. Multi-response optimization of germination conditions allowed to identify the most suitable germination conditions for maximizing nutritional and bioactive quality of sprouted oat flours (16 °C-216 h and 18 °C-156 h for hulled and dehulled varieties, respectively). This study provides useful information regarding the production of gluten-free flours from sprouted oat with improved nutritional and healthy attributes.