Greenhouse tomato production faces multiple challenges, including the excessive use of nonrenewable substrates that are difficult to dispose of after use. Currently, most growers propagate tomatoes in rockwool, but there is an increasing demand for sustainable media. The objective of this research was to evaluate sustainable and organic alternatives for greenhouse propagation of tomato seedlings intended for high-wire production. Different organic and inorganic substrates were evaluated in three experiments, using a nutrient solution composed of a complete water-soluble fertilizer. Germination and growth parameters, including height, stem diameter, number of leaves, leaf area, foliar chlorophyll levels (SPAD), and shoot fresh and dry weight, were measured. In the first experiment, which employed overhead irrigation, rockwool, coir, wood fiber–coir mix, medium-grade pine bark, pine bark < 0.64 cm, and pine bark < 0.32 cm were evaluated. Tomato germination was faster and achieved higher percentages with pine bark < 0.64 cm compared to other substrates. However, growth performance was similar or better in coir than in rockwool four weeks after transplantation. For the second experiment with sub-irrigation only, rockwool, coir, wood fiber–coir mix, pine bark < 0.32 cm bark, and peat were evaluated at different container heights. Peat resulted in greater growth across all parameters, followed by wood fiber–coir mix in all container heights, while pine bark had the least growth across all measured parameters. In the third experiment with overhead irrigation, rockwool, wood fiber–coir mix, pine bark < 0.32 cm, and a commercial peat-based mixture were evaluated under different fertilizer rates (electrical conductivity of 1.1 and 2.2 mS·cm−1). Wood fiber–coir mix, peat-based mix, and rockwool were the substrates with the highest values for all evaluated parameters. While all the organic substrates showed potential for use in tomato propagation, pine bark < 0.32 cm bark and wood fiber–coir mix provided the best media for germination. Peat and wood fiber–coir mix showed the best media for subsequent seedling growth and demonstrated potential to be used as substitutes for rockwool.