This study aimed to investigate the chemical composition and biological potential of needle extracts from five pine species, including antimicrobial, antioxidant, and cytotoxic activity, as well as their influence on cell cycle progression. Needle extracts were prepared using three extraction methods: conventional maceration (CM), ultrasound-assisted extraction (UAE), and digestion (D). The chemical profile was determined with an emphasis on phenolic acids, flavonoids, and related phenolic compounds. The highest total phenolic content was observed in Pinus sylvestris (3.438 mg/g GAE), followed by Pinus heldreichii (2.732 mg/g GAE). Rutin, ferulic acid, and quercitrin were identified as the predominant phenolic compounds. The highest total flavonoid content was found in Pinus pinea extracts obtained by digestion (1.213 mg/g QE), followed by P. heldreichii (1.074 mg/g QE) and Pinus halepensis (1.074 mg/g QE), both obtained by UAE. Among all examined species, Pinus pinea exhibited the highest TTC values, regardless of the extraction method (7.31–8.21 mg/g GAE). Antibacterial testing showed that P. pinea had an MIC of 19 mg/mL against Enterococcus faecium, while P. sylvestris had the same MIC against Bacillus spizizenii. All extracts exhibited cytotoxic effects using MTT assay against HeLa cells at concentrations of 8%, 16%, and 32%, while LS 174T cells were the least sensitive. Pine needle extracts from Montenegro are a valuable source of phenolic and flavonoid compounds, and they demonstrate antimicrobial and cytotoxic activities. The results support the need for further in vivo studies and elucidation of mechanisms of action in order to assess their potential application as novel bioactive agents.
