The potential of biochar to improve crop productivity has received interest in recent years; however, little is known about the effects of biochar on crop nutritional quality. In this study, effects of three different biochars (wheat straw biochar, poplar biochar and olive residues biochar) were determined on the major fruit-size parameters, physico-chemical and nutritional properties of tomato (Lycopersicon esculentum L.) cv. Rio Grande. Application of biochar alone was sufficient to sustain the fruit growth, but results were affected by feedstock source of biochar. There were no significant differences in size and weight parameters and the sugar content was not significantly modified by biochar amendment. On the other hand, secondary metabolites showed changes relating to biochar type. Total phenol and flavonoid contents, as well as antioxidant activity, were higher in fruits grown in substrate amended with straw biochar and olive residues biochar. Lycopene, β carotene and lutein concentrations from tomato fruits grown on substrates amended with different biochars were significantly lower than from the control. The data require confirmation in field experiments; however, this study offers new knowledge about the biochar effects on horticultural crops.