This study aims to evaluate the interspecific variation in polyphenolic profiles and biological activities of acorn flours from three native Tunisian Quercus L. species (Q. ilex L., Q. suber L., and Q. canariensis Willd.). Q. canariensis extracts are the richest in total phenolic, flavonoid and hydrolysable tannin contents. Six phenolic compounds were identified by HPLC-DAD analysis. Chlorogenic acid was the dominant compound in Q. canariensis and Q. ilex acorns. Whereas in Q. suber, caffeic acid was the main component and, along with trans-ferulic acid, was exclusive to this species. Hyperoside was notably identified in Q. canariensis. The UHPLC-DAD-MS analysis of hydrolysable tannins revealed twelve compounds. Acorns of Q. canariensis and Q. suber were dominated by ellagitannins, whereas Q. ilex contained mainly gallotannins. To our knowledge, these compounds are identified for the first time in Tunisian acorns. Q. canariensis exhibited the strongest antioxidant potential with DPPH, ABTS and FRAP assays, as well as the highest antibiofilm and anti-α-amylase activities. All extracts inhibited ATCC pathogenic bacterial strains while largely sparing the beneficial probiotic Limosilactobacillus fermentum. This result indicates a selective antibacterial effect not previously reported for Quercus acorns. Q. canariensis may represent a potential source of functional food ingredient, nutraceuticals, and pharmaceuticals, which remains to be confirmed through in vivo investigations.