The use of biotechnological systems to enhance the synthesis of high-value metabolites represents a key strategy to promote sustainability. The aim of this study was to evaluate the effect of different stress inducers (50 µM methyl jasmonate, 100 µM salicylic acid, 3% glycerol, and elevated temperature at 35 °C) on lipid production in callus cultures of Theobroma cacao L., Nacional variety (fino de aroma), a species of high economic and cultural value for Ecuador. Calli were established in MS, B5, and WPM media, supplemented with specific hormonal combinations (2,4-D, NAA, KIN, and BAP). WPM medium enriched with NAA (2 mg/L) and KIN (0.5 mg/L) promoted the highest biomass accumulation. From the biomass generated under these optimal conditions, the different stress inducers were applied. After a 28-day incubation period, calli were collected and subsequently subjected to acid hydrolysis, followed by lipid extraction using the AOAC 991.36 method. The treatment with methyl jasmonate resulted in the highest concentration of lipid compounds (25.6 mg), followed by salicylic acid (14.6 mg), indicating metabolic activation without compromising cell viability. Glycerol and elevated temperature caused oxidative stress with variable responses. FTIR spectroscopy confirmed the presence of saturated and unsaturated fatty acids, showing a significant increase in total lipid concentration among treatments. Salicylic acid promoted the highest accumulation of saturated fatty acids (10.4 mg/mL), followed by methyl jasmonate (8.1 mg/mL), suggesting differential activation of lipid biosynthetic pathways. These results demonstrate that applying controlled stress can modulate the production of target compounds in plant systems, offering a sustainable alternative for cacao valorization.