<p>The complex interplay among diet, environment, and physiology in mesopelagic fishes is generally poorly understood. In this study, we analyzed the fatty acid composition of 16 mesopelagic fish species across four families from the Tasman Sea. Significant interspecific differences in fatty acid trophic markers (FATMs) were found, indicating diverse feeding strategies and ecological roles. <i>Cubiceps caeruleus</i> exhibited the highest relative levels of long-chain polyunsaturated fatty acids (≥ C₂₀, LC-PUFAs), suggesting a lower trophic position, while members of the Myctophidae family showed elevated levels of long-chain monounsaturated fatty acids (LC-MUFAs), consistent with copepod feeding. Biological and environmental factors further influenced FATMs, with significant correlations observed between fish size and general carnivory markers across the mesopelagic fish assemblage. These findings highlight how species-specific traits and environmental conditions influence the feeding ecology of mesopelagic fishes. By explicitly linking dietary composition to both intrinsic and extrinsic drivers, this study provides new insights into the functional role of mesopelagic fishes in carbon and energy transport, thereby offering valuable inputs for climate and ecosystem models and resource managers.</p>