Substrate composition plays a vital role in the soilless cultivation of Helleborus; high-quality substrates can create optimal growth conditions and enhance plant quality. However, knowledge regarding suitable substrates for Helleborus remains limited. The aim of the study was to test the effects of different substrates on growth of potted Helleborus × hybridus seedlings. In total, 12 treatments were formulated by mixing organic components (moss peat, cocopeat, domestic peat) with inorganic particles (perlite, kanuma soil, vermiculite) at a 1:1 ratio, using moss peat moss as the control. The results indicated that substrates of moss peat + vermiculite (1:1) and cocopeat + vermiculite (1:1) significantly promoted plant height, shoot number, leaf number, and root development. The treatment with domestic peat + perlite + kanuma soil + vermiculite (3:1:1:1) yielded the highest soluble protein content, whereas the CK group showed the highest soluble sugar content. A comprehensive evaluation by Principal Component Analysis (PCA) identified moss peat + vermiculite (1:1) as the optimal substrate, followed by coir + vermiculite (1:1). Considering economic costs and environmental protection factors, the cocopeat + vermiculite (1:1) mixture demonstrated superior potential. Collectively, our study clarifies the effects of different substrate compositions and provides new insights for achieving cleaner and more efficient soilless cultivation of Helleborus.