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Biostimulants can enhance horticultural crop production. However, their application in hydroponically grown lettuce is still limited, and information regarding their relative efficacy is lacking. A greenhouse trial was conducted to address this issue. Five nutrient solution treatments were evaluated on two lettuce cultivars: butterhead and red oak-leaf. The treatments included a half-strength modified Hoagland solution (Hs-H); a full-strength modified Hoagland solution (Fs-H); and Hs-H supplemented with 50 mg L−1 fulvic acid (FA), 334 mg L−1 seaweed extract (SE), or 5 mL L−1 gamma polyglutamic acid (PGA). The results indicated that the shoot biomass observed after biostimulant supplementation was significantly greater than or comparable to that observed with Fs-H. Nutrient solutions supplemented with SE and PGA led to a greater increase in the root biomass than that realized with Hs-H and Fs-H treatments. The Hs-H + FA treatment resulted in the lowest root-to-shoot ratio on a fresh weight basis among all treatments. The nitrate concentration in the shoot was significantly reduced following biostimulant supplementation compared to that realized with Fs-H and Hs-H treatments. Nutrient solutions supplemented with SE and PGA also decreased soluble sugar concentrations compared to that achieved using Hs-H and Fs-H treatments. FA and SE improved nutrient uptake for both cultivars, but PGA had a minimal effect on nutrient uptake. The two cultivars varied in their responses to biostimulant supplementation with regard to biomass, quality traits, and nutrient uptake. This study supports using fulvic acid and seaweed extract, rather than γ-PGA, in hydroponic lettuce production systems.