This study provides the first comprehensive evaluation of the bioactive potential of acorn flour extracts (Quercus robur, Fagaceae) prepared at different temperatures (20, 60, 80 and 100 °C), focusing on polyphenolic content, antioxidant properties and enzyme inhibitory activities. Through HPLC-ESI-MS/MS analysis, 36 bioactive compounds were identified, with the extract at 60 °C showing the highest concentrations of key polyphenols, notably gallic acid (210,008.9 mg·kg−1) and ellagic acid (45,469.6 mg·kg−1). This extract also exhibited a high antioxidant activity and significant inhibition of glucosidase and acetylcholinesterase, suggesting potential benefits for diabetes management and neuroprotection. The results indicate that extraction temperature affects bioactivity, with the 60 °C extract standing out as a promising candidate for nutraceutical, pharmaceutical, and cosmeceutical applications due to its rich polyphenol profile and potent biological properties.