The effect of a new soil substrate (hazelnut husk + gyttja) on the acclimatization success of in vitro-produced plantlets of four ornamental species (Rosa hybrida, Loropetalum chinense, Aloe vera, Hylocereus undatus) was assessed. Phytotoxicity test results for all the combination of hazelnut husk + gyttja substrates studied was similar to the standard cocopeat mixture used as control. Mortality rate of acclimatized plantlets on either hazelnut husk or gyttja substrates or standard cocopeat were higher for woody species Rosa hybrida and Loropetalum chinense compared to the succulent species Aloe vera and Hylocereus undatus. Findings of this study demonstrated that incorporation of gyttja into the hazelnut husk at a rate of 2% and 5% had similar positive effects on survival rate, plant height, shoot, and root dry biomass, and Dickson quality index for all tested plant species, with to the cocopeat control plants. Therefore, hazelnut husk and gyttja could be successfully used as soil substrate for ex vitro acclimatization, after preliminary tests for the specific plant species.