The biochar applied to the soil create complex interactions that impact the plant development and soil quality. The search for alternatives to decrease the use of substrates of limited use has been a challenge for science and agricultural producers. The use of biochar from different waste sources and the relationship between seed germination and plant parameters show potential; however, there is need for a better understanding of these factors. Although the potential benefits of biochar, such as improved plant growth and seed germination rates, have been demonstrated, there is still a need for a deeper understanding of the factors influencing these effects. Here, we investigated the use of biochar from different sources as alternative substrate in the production of lettuce and tomato seedlings. To test this hypothesis, seed germination, plant growth, and soil parameters were assessed using two biochar sources (poultry litter-PL, and viticulture waste-VW) at different doses [1% (1), 2.5% (2.5), and 5% (5)]. Our study shows that both biochar sources, mainly PL, promoted the germination and initial development of lettuce plants, similar to commercial substrate; however, they showed no effects on tomato. Moreover, the multivariate analysis revealed that the commercial substrate and PL1 are correlated with most vectors of lettuce plant parameters. In contrast, the commercial substrate and PL5 are in the same quadrant and clustered along with most vectors of tomato plant parameters, proving to be the most efficient for initial plant development. Overall, our findings expand the knowledge on waste reuse transformed into biochar as an effective alternative component of substrate for the production of lettuce seedlings, mainly biochar from PL.