The use of biostimulants in modern agriculture has rapidly expanded in recent years, owing to their beneficial effects on crop yield and product quality, which have come under the scope of intensive research. Accordingly, in the present study we appraised the efficacy of two plant-derived biostimulants, the legume-derived protein hydrolysates Trainer®® (PH), and the tropical plant extract Auxym®® (TPE) on two lettuce cultivars (green and red salanova®®) in terms of morpho-physiological and biochemical traits (primary and secondary metabolites). The two cultivars differed in their acquisition capacity for nitrate and other beneficial ions, their photosynthetic and transpiration rates, and their ability to synthetize and accumulate organic acids and protective metabolites. The biostimulant effect was significant for almost all the parameters examined but it was subjected to significant cultivar × biostimulant interactions, denoting a cultivar-dependent response to biostimulant type. Notwithstanding this interaction, biostimulant application could potentially improve the yield and quality of lettuce by stimulating plant physiological processes, as indicated by the SPAD index (leaf chlorophyll index), ACO2 (assimilation rate), E (transpiration), and WUEi (intrinsic water use efficiency), and by increasing concurrently the plant mineral content (total N, K, Ca, Mg) and the biosynthesis of organic acids (malate, citrate), phenols (caffeic acid, coumaroyl quinic acid isomer 1, dicaffeoylquinic acid isomer 1), and flavonoids (quercetin-3-O-glucuronide, quercetin-3-O-glucoside). Biostimulant action may facilitate the bio-enhancement of certain lettuce cultivars that are otherwise limited by their genetic potential, for the accumulation of specific compounds beneficial to human health.