The pungency of chili peppers is conferred by compounds called capsaicinoids that are produced only in the fruits of the Capsicum genus. Accumulation of capsaicinoids in these fruits may be affected by environmental conditions such as water and nutrient stresses, although these effects may vary even among genotypes within a species. The Habanero pepper (Capsicum chinense Jacq.), grown in the Yucatán, is in especially high demand as a result of its unique flavor, aroma, and pungency and is the second most important commercial crop in the state after the tomato. Although the Habanero pepper is a significant economic resource for the region, few studies have investigated the effects of abiotic stresses on capsaicinoid production. In this study, the effects of water stress on plant growth, capsaicinoid accumulation, and capsaicin synthase activity were evaluated. Habanero pepper plants under water stress had a lower height, root dry weight, and root/shoot relation than control plants, which were irrigated daily. However, fruit growth and production were unaffected by water stress. Capsaicin and dihydrocapsaicin concentrations increased in fruits of stressed plants compared with control plants, and this effect was correlated with fruit age. However, capsaicin synthase activity was reduced in response to water stress, and this effect depended on both stress severity and fruit age. These results provide new information on the regulation of capsaicinoid metabolism in response to abiotic stress from the fruit of a highly pungent chili pepper.