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This study ascertained the accumulation of polyprenol from four Irish conifer species Picea sitchensis, Cedrus atlantica ‘Glauca’, Pinus sylvestris and Taxus baccata and one flowering tree Cotoneaster hybrida using supercritical fluid extraction with carbon dioxide (SFE-CO2) and solvent extraction. The effects of SFE-CO2 parameters such as temperature (ranged from 40 to 70 $$^\circ{\rm C}$$ ∘ C ), pressure (ranged from 100 to 350 bars) and dynamic time (from 70 min to 7 h) were analysed on the extraction efficiency of polyprenol. Qualitative and quantitative analysis of polyprenol was examined using high-performance liquid chromatography. Results showed that P. sylvestris accumulated the highest polyprenol yield of 14.00  ± $$0.4$$ 0.4 mg g−1 DW when extracted with hexane:acetone (1:1 v/v). However, with SFE-CO2 conditions of 200 bars, 70 $$^\circ{\rm C}$$ ∘ C , 7 h, with absolute ethanol as a cosolvent with a flow rate of 0.05 ml min−1, P. sitchensis accumulated the highest polyprenol yield of 6.35 ± $$0.4$$ 0.4 mg g−1DW. This study emphasised the potential application of SFE-CO2 in the extraction of polyprenol as an environmentally friendly method to be used in pharmaceutical and food industries.