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Raspberry fruits are a valuable source of bioactive compounds. The study used the modification of the substrate (coconut fibre), consisting of the use of various organic and mineral additives, in the soilless cultivation of raspberries. The additives influenced the biosynthesis of bioactive compounds in the raspberry fruits by modifying the sorption properties and the abundance of the substrate. The influence of the additives on the content of polyphenols was determined as well as their profile (UPLC-MS), antioxidant potential (ABTS), vitamin C content, and the activity of selected enzymes that are markers of stress and resistance to abiotic factors. In the study, a significant effect of these additives was observed on the biosynthesis of polyphenols in raspberry fruit. The highest increase in the content of these compounds in relation to the control sample (substrate—100% coconut fibre), namely 37.7%, was recorded in the case of fruit produced on coconut substrate enriched with sheep wool. These fruits were also characterised by a significantly different profile of these compounds. These changes were caused by readily available ammonium nitrogen and free amino acids in the decomposition of proteins contained in the sheep wool. This was confirmed by the recorded content of chlorophyll SPAD in the plant leaves and the activity of selected enzymes, which proves a low level of stress and good condition of the plants. [ABSTRACT FROM AUTHOR]