Due to the increase in aging populations and the prevalence of aging-associated diseases, there is a growing interest in finding therapeutic interventions. Antioxidants play a vital role in mitigating the adverse conditions associated with aging. In this study, we investigated the impact of Dendrobium officinale alkaloids (DOA) cultivated in diverse environments on antioxidative and anti-aging responses in Caenorhabditis elegans. Three distinct sources of DOA, represented as tree (TR)-DOA, greenhouse (GH)-DOA, and rock (RK)-DOA, were examined. Following initial testing of three DOA concentrations, 10 µg/mL was selected, which increased the TJ1060 lifespan by 23%, and exhibited no toxicity. TR-DOA, GH-DOA, and RK-DOA exhibited robust antioxidative effects, significantly reducing reactive oxygen species levels by 37%, 54%, and 60%, respectively on day 1, and 68%, 73%, and 75% on day 5, respectively. On day 4, TR-DOA, GH-DOA, and RK-DOA significantly reduced lipofuscin levels by 36%, 51%, and 39%, respectively (P 0.05, respectively). RK-DOA, GH-DOA, and TR-DOA delayed paralysis (P < 0.0001, in all cases) and significantly increased worm activity (P < 0.001, P < 0.001, and P < 0.01, respectively). RK-DOA, GH-DOA, and TR-DOA also significantly increased the rate of body bend on day 5 (P < 0.001, P < 0.01, P < 0.0001) and day 10 (P < 0.001, P < 0.0001, P < 0.01). Our findings suggest that DOA, particularly RK-DOA, offers promising antioxidative and anti-aging benefits in C. elegans. The differential responses among DOA variants highlight the importance of the cultivation environment in shaping the bioactivity of natural compounds.