The quality of tomato fruits is influenced by preharvest factors. Trichoderma are considered biostimulants with potential to improve growth and development in plants, as well as the quality of the fruits. The objective of this study was to evaluate the effect of foliar application of Trichoderma on the quality of tomato fruits at different cluster levels on the same plant in two commercial hybrids using three different substrates in a greenhouse. Six foliar applications of a liquid biological preparation of Trichoderma were performed at a dose of 4 mL L−1 every 15 days after transplanting. The results show that the foliar application of Trichoderma increased the ratio of soluble solids to titratable acidity of the fruits at different cluster levels, which was mainly due to the decrease in acidity rather than the increase in soluble solids. The decrease in titratable acidity is in accordance with the increase in the pH of the fruits. Trichoderma have a positive effect on titratable acidity, pH and electrical conductivity of fruits. In the Cid hybrid grown in the tezontle substrate, the application of Trichoderma increased the ratio of total soluble solids to titratable acidity. At the same time, sand increased the percentage of juice in the fruits. Foliar application of Trichoderma increases the quality of tomato fruits, and as such, should be considered as a crop management option.