The main goal of the study was to examine and compare the inhibition effect of knotwood extractives of Scots (Pinus sylvestris) and black pine (Pinus nigra) against the two white-rot fungi and two brown-rot fungi. Knotwood was extracted in a Soxhlet apparatus. Extracts were chemically analyzed, and the inhibitory effect of purified pinosylvins and crude hydrophilic extracts was tested against growth of the white-rot fungi Schizophyllum commune and Trametes versicolor and the brown-rot fungi Fibroporia vaillantii and Gloeophyllum trabeum with the in vitro antifungal assay. Knotwood of Scots pine and black pine contained comparable amounts of total extractives. Pinosylvin, pinosylvin monomethyl ether and nortrachelogenin were characteristic compounds in knotwood extracts of both pines. Scots pine knotwood contained larger amounts of pinosylvin than black pine. The ratio between pinosylvin monomethyl ether and pinosylvin was higher in black pine knotwood. Purified pinosylvins and crude hydrophilic extracts inhibited the growth of all the white-rot and brown-rot fungi tested. Pure pinosylvins better inhibited fungal growth than crude knotwood extracts, whereas the efficiency was not unambiguous and relates to fungi species. Crude hydrophilic extracts of Scots pine more efficiently inhibited fungal growth than extracts of black pine. With the present investigation, hydrophilic extracts of pine knotwood were demonstrated as formulations of natural compounds with good antifungal properties. Broken tops of Scots pine, which can lie on forest grounds for a month or even more, still contain high amounts of phenolic extractives and are therefore a potential raw material for recovery of bioactive compounds.