Biochar applicability as culture medium amendment is rarely investigated due to its uncovered impact on in vitro cultured plants. This study assessed the feasibility of several biochars as alternatives to activated charcoal, for micropropagation of two ornamental Daphne species (Thymelaeaceae). We distinguished metabolic responses that were specific for: a) medium supplementation with any type of charcoal; b) biochar addition; c) biochar activation; d) the process of charcoal activation itself. We compared impacts of industrially produced biochar and three different biochars made in small-scale stoves (from residues of Gliricidia sepium) on organogenic capacity and physiological status in Daphne sp. shoots. Metabolite profiling revealed that biochars differently modulated accumulation of endogenous phytohormones and osmolytes in the shoots. Biochars reduced accumulation of proline, betaines and stress-related phytohormones (ABA, jasmonates), and impacted carbohydrate profile. For D. jasminea the most impactful was biochar activation, which increased micropropagation efficiency by elevating cytokinin and soluble sugars content. For D. tangutica charcoal/biochar activation, and application of biochar reduced accumulation of ABA and jasmonic acid, increased level of gibberellins, salicylic and benzoic acid, and altered carbohydrate composition. The study revealed that tested biochars can be used as a sustainable medium supplements without negative impact on production of new microshoots. Based on studied species we showed that biochar supplements could stimulate plantlet formation (D. jasminea,) ameliorate stress response (D. tangutica), and constitute a way of undesired biomass utilization (invasive species G. sepium).