The reuse of substrates for more than one growing season is an excellent alternative to reduce production costs and to improve the sustainability of the ornamental plant production system. The objectives of the present trial were to study the effects of substrate reuse on Lilium var. Helvetia plants cultivated in shipping crates in a polyethylene greenhouse, as well as possible changes in the physical and physical–chemical properties of the substrates employed. Peats used during two and three cultivation cycles and new unused peat were tested. The reused substrate reduces the particle fraction with higher diameter size, porosity, and organic matter and increases particle bulk and substrate carbon (C) / nitrogen (N) ratio, due to the substrate decomposition. However, it does not affect flowers, flower stems and leaves, and dry and fresh matter. The level of phosphorus (P) and magnesium (Mg) in leaf, as well as nitrate, potassium (K), and Mg in the substrate solution are similar among the three kinds of substrates studied. Nevertheless, the substrate used for three cultivation cycles leads to increases in organic N and K and decrease of calcium in leaves.