Callunene, a natural component of heather (Calluna vulgaris) nectar, has previously been shown to protect bumblebees from infection by the trypanosomatid Crithidia bombi. Here, we demonstrate that callunene exhibits antiparasitic activity against several trypanosomatid species, including Crithidia bombi, Leishmania mexicana, and Trypanosoma brucei. Notably, callunene’s in vitro efficacy against T. brucei was comparable to that of nifurtimox, although its cytotoxicity toward human cells may limit direct therapeutic application. Using a biotinylated callunene analog in the pull-down assay, we identified NIPSNAP, a mitochondrial protein involved in mitophagy regulation, as a primary molecular target of this compound in C. bombi. Moreover, callunene alters acidocalcisome abundance, further connecting its role to regulation of mitochondrial physiology. Given its effects on mitochondria and ability to interact with NIPSNAP, callunene represents a promising chemical probe for studying mitophagy, a poorly understood process in trypanosomatids, and may provide new insights into mitochondrial biology of these parasites.