Improper agricultural waste management harms the environment. Certain agricultural waste materials, such as oil palm kernel wastes, can be used as soil amendments or fertilizers. This study aimed to explore their potential as an alternative hydroponic substrate. The effects of different hydroponic substrates on lettuce metabolite production, specifically biochar, have not been previously studied. The study compared the bioactive properties and metabolite profiles of red lettuce grown under various treatments: T1 (control; hydroton), T2 (perlite), T3 (palm kernel biochar), T4 (hydroton + palm kernel biochar), and T5 (perlite + palm kernel biochar). Lettuce grown with T4 exhibited the highest chlorophyll contents, while T3 resulted in the highest carotenoid, anthocyanin, phenolic, and flavonoid contents. T3 demonstrated the highest DPPH radical scavenging activity and ferric reducing potential, while T4 exhibited the highest ABTS radical-scavenging activity. LC-MS-QTOF analysis tentatively annotated a total of 38 metabolites, with 12 compounds identified based on databases and literature search. These compounds cluded phenolic and non-phenolic compounds, as well as additional classes like coumarins, xanthine alkaloids, and glycosides. Red lettuce grown in T3 showed the highest number of metabolites (18), followed by T5 (15), T2 and T4 (13), and T1 (6). E-Suberenol, a potent antioxidant, displayed a strong correlation with the treatments using palm kernel biochar. This study suggests that palm kernel biochar can serve as a cost-effective, renewable, and sustainable alternative hydroponic substrate.