Commercial forest plantations in Chile are dominated by pine (Pinus radiata) and eucalyptus (Eucalyptus globulus). Tree bark is the main by-product of the forestry industry and has low value, but great potential for use as an agricultural substrate. However, the direct use of bark fibers may cause plant phytotoxicity due to the presence of polyphenolic and other compounds. This study aims to evaluate the physicochemical properties of E. globulus and P. radiata bark after water extraction treatments. The phytotoxicity of the resulting extracted bark alone and that mixed with commercial substrates (coconut fiber, moss, peat, and composted pine) at different ratios (25 to 75 wt%) were assessed using the Munoo-Liisa vitality index (MLVI) test. For all treatments, the seed germination and growth of radish (Raphanus sativus) and Chinese cabbage (Brassica rapa) species were evaluated and compared to a commercial growing medium (peat) as a control. The optimal mixture for seed growth was determined to be 75% extracted E. globulus bark fiber and 25% commercial substrates such as peat (P), coconut fiber (C), moss (M), and composted pine (CP), as indicated by the MLVI and germination results. Two phytostimulant products, chitosan and alginate-encapsulated fulvic acid, were added to the best substrate mixture, with the purpose of improving their performance. Encapsulated fulvic acid at 0.1% w/v was effective in promoting plant growth, while chitosan at all of the concentrations studied was effective only for mixture 75E-25CP. The mixture of E. globulus fiber and commercial substrates, containing a high proportion of water-extracted fiber (75%), shows the potential to be used in the growth of horticultural crops and in the plant nursery industry.