Pinus species are notable in Mediterranean regions due to their ecological and economic importance. Various parts of these species are widely used in traditional medicine, especially pinecones, which are a significant source of bioactive compounds. The current study aimed to evaluate the phytochemical composition and biological properties of the aqueous extracts obtained by maceration from three Pinus petal fractions, from P. halepensis Mill., P. brutia Ten., and P. pinea L. (APW, BPW, and PPW respectively), and the core fractions of the same species (ACW, BCW, and PCW respectively). The results showed that APW demonstrated superior performance compared to other species and fractions (p ≤ 0.05), with the highest total polyphenol content (203.51 mg GAE/g DW) and the highest antioxidant potential (IC50 = 13.51 µg/mL) against DPPH free radical. All extracts showed high anticancer activity against HeLa and HepG2 cancer cell lines, and low inhibition against HEK-293, a normal cell line (<15%), indicating that none of extracts have any toxicity effect. Furthermore, only APW exhibits a significant inhibition against α-glucosidase with 77.20% at 50 µg/mL. HPLC-DAD analysis was conducted to identify 14 compounds. GC-MS analysis was conducted to identify 28 compounds, of which 11 were detected for the first time in this species. This study offers valuable insights into phytochemistry and potential therapeutic applications of pinecones.