The production of lettuce has increased significantly due to the use of hydroponic systems that rely on substrates. Disposal and acquisition costs present problems, necessitating the identification of sustainable alternatives. The present study aimed to evaluate the use of Eichhornia crassipes (water hyacinth) dry matter in a substrate for the formation of lettuce seedlings. Water plants were collected to obtain their dry matter, and twelve mixtures were formed with Sphagnum and perlite. Mixtures with more water hyacinth dry matter exhibited greater water retention. However, these mixtures also lost water at a faster rate than those containing primarily Sphagnum dry matter did. Higher percentages of germination were detected in the mixtures with water hyacinth dry matter, but these seedlings also presented higher concentrations of proline, such as 16.0 µg mL−1. The mixtures with water hyacinth dry matter presented the highest ion concentrations, mainly at high levels of humidity. Mixtures with a high proportion of water hyacinth dry matter had a greater water retention capacity and a high percentage of lettuce seed that germinated. The mixtures with a higher proportion of Sphagnum led to greater root length, greater concentrations of chlorophyll in cotyledonary leaves, and better morphological development of the seedlings.