Lettuce (Lactuca sativa L.) is a leafy vegetable cultivated widely for its fast and year-round production and its beneficial phytochemical content, which may be boosted further by plant biostimulants that are considered eco-sustainable means for enhancing horticultural crop production. A greenhouse experiment was carried out to evaluate the yield and qualitative parameters of two differently pigmented lettuce cultivars grown in a floating raft system either untreated or treated (leaf, root or leaf/root application) with vegetal protein hydrolysates (PHs). For foliar application (F), lettuce plants were sprayed at a dose of 3 mL L−1, whereas for root application, 0.15 (T1) or 0.3 (T2) mL L−1 was applied to the nutrient solution alone or in combination with foliar spray (T1 + F and T2 + F) with the same foliar concentration. Bio-morphometric and production data were collected after harvest. Physiological and plant nutrition assays included leaf gas exchange, leaf fluorescence, SPAD index, mineral content, carotenoids, total phenols, total ascorbic acid content and antioxidant activities. Cultivar-specific reactions to biostimulant application were noted: whilst the green pigmented cultivar thrived under nutrient solution applications and recorded higher yield by 82.7% (T1) or (T1 + F) and 71.7% (T2), the red cultivar thrived under combined treatments, yielding 55.4% (T2 + F) higher than control and providing the most concentrated phytochemical content. These latter treatments also engendered the highest SPAD index, Fv/Fm ratio, CO2 assimilation, stomatal conductance and transpiration. In addition, the T2 + F treatment boosted ‘Canasta’ hydrophilic antioxidant activity (21.9%) and total ascorbic acid (5.6-fold). Nutrient solution treatments alone proved advantageous when compared to foliar treatments, while mixed treatments proved genotype-specific. New research on genotype specificity of biostimulant effects is warranted for future use, in order to rationalize biostimulant application modes and dosages.