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Medicinal plant cultivation in Controlled Environment (CE) is increasing in the context of the new findings concerning the abiotic stress factors manipulation that leads to a significant increment in bioactive substances. Pathogen control is a vital part of the cultivation system, therefore the study was focused on biological methods of controlling a frequently occurring disease, by inoculating the sterile substrates specific in hydroponics, with the beneficial organism. Hypericum perforatum seedlings were inoculated with Fusarium oxysporum and with Trichoderma spp. The results showed that the method of inoculation had a significant effect on the success of the biological control. The plants treated with Trichoderma spp. had a two-fold increase in foliar biomass and better development of roots than the plants inoculated with the pathogen. Morphologically there were no significant differences, with some notable exceptions. The health status of the seedlings inoculated with the pathogen showed signs of the disease, even in the presence of Trichoderma spp. Finally, determinations of Volatile Organic Compounds (VOCs) at the root level showed that the biotic stress was considerable higher in the rockwool substrate that increased the secondary metabolism giving new perspectives in the cultivation of medicinal plants in CEA.