The genus Trichoderma is widely used in agriculture as a biological agent and biofertilizer, enhancing crop yield and quality. However, its use in hydroponic systems is limited. This study evaluated the potential of Trichoderma asperellum as a growth promoter for lettuce (Lactuca sativa L.) cv. Starfighter RZ in a floating-root hydroponic system (FHS). T. asperellum strains (TaMFP1 and TaMFP2) were isolated from soil and identified morphologically and molecularly. The experiment used a completely randomized design with the following four treatments (n = 4): root spraying with TaMFP1, TaMFP2, T. harzianum (Trichospore(®)), and uninoculated plants (control). After 30 days, morphological, biochemical, and quality parameters were analyzed. All Trichoderma treatments significantly increased plant height (19.0%), root length (25.7%), total fresh biomass (76.4%), total dry biomass (82.63%), and number of leaves (18.18%). The nitrate levels in leaves were unaffected by TaMFP1 and TaMFP2, while Trichospore(®) reduced the nitrate content by 24.94%. The foliar nitrogen content increased with specific treatments, though the phosphorus and magnesium levels decreased. Visual quality traits, including appearance and firmness, remained unchanged. T. asperellum strains TaMFP1 and TaMFP2 enhanced vegetative growth without compromising quality, demonstrating their potential as sustainable tools for hydroponic lettuce production in controlled environments.