AbstractBACKGROUNDProtein hydrolysates (PHs) can enhance plant nitrogen nutrition and improve the quality of vegetables, depending on their bioactive compounds. A tomato greenhouse experiment was conducted under both optimal (14 mM) and suboptimal (2 mM) nitrogen (N‐NO₃) conditions. Tomatoes were treated with a new Malvaceae‐derived PH (MDPH) and its molecular fractions (MDPH1, >10 kDa; MDPH2, 1–10 kDa and MDPH3, <1 kDa).RESULTSUnder optimal N conditions, the plants increased biomass and fruit yield, and showed a higher photosynthetic pigment content in leaves in comparison with suboptimal N, whereas under N‐limiting conditions, an increase in dry matter, soluble solid content (SSC) and lycopene, a reduction in firmness, and changes in organic acid and phenolic compounds were observed. With 14 mM N‐NO₃, MDPH3 stimulated an increase in dry weight and increased yield components and lycopene in the fruit. The MDPH2 fraction also resulted in increased lycopene accumulation in fruit under 14 mM N‐NO₃. At a low N level, the PH fractions showed distinct effects compared with the whole MDPH and the control, with an increase in biomass for MDPH1 and MDPH2 and a higher pigment content for MDPH3. Regardless of N availability, all the fractions affected fruit quality by increasing SSC, whereas MDPH2 and MDPH3 modified organic acid content and showed a higher concentration of flavonols, lignans, and stilbenes.CONCLUSIONThe molecular weight of the peptides modifies the effect of PHs on plant performance, with different behavior depending on the level of N fertilization, confirming the effectiveness of fractioning processes. © 2024 Society of Chemical Industry.