Root health disorders are among the primary constraints in hydroponic lettuce cultivation, especially under conditions of suboptimal aeration, high temperatures, and fluctuating humidity. Hydrogen peroxide (H2O2) is commonly used to increase dissolved oxygen availability, suppress certain pathogens, and promote root health. This study aimed to evaluate the response of four lettuce varieties grown in a Nutrient Film Technique (NFT) hydroponic system with the application of 3% H2O2 and to identify the variety with the best agronomic performance. The research was conducted from October to December 2023 at Kebun Sayur 24 Hidroponik, Bojongsari Village, Alian District, Kebumen Regency. The experiment was arranged in a nonfactorial completely randomized design with four varieties—RZ Junction, Romaine, Mondai, and Rex—each replicated six times. The observed variables included plant height, root length, number of leaves, fresh plant weight, dry plant weight, fresh root weight, dry root weight, and leaf area at harvest, 45 days after sowing. Data were analyzed using analysis of variance followed by the 5% Honestly Significant Difference test. The results showed that varietal differences had a highly significant effect on plant height, number of leaves, dry plant weight, fresh root weight, dry root weight, and leaf area, but had no significant effect on root length and fresh plant weight. Romaine showed the best performance in plant height (14.37 cm), fresh root weight (9.17 g), dry root weight (0.66 g), and leaf area (671 cm²), whereas Rex produced the highest number of leaves (15.50). Therefore, Romaine was the most promising variety for NFT hydroponic lettuce cultivation under 3% H2O2 application.