Holothurians are the dominant megabenthic deposit feeders in the Peru Basin (South-East Pacific) and feed to various degrees of selectively on the heterogenous pool of sedimentary detritus, but diet preferences for most holothurian species are unknown. This study reconstructs the diets of 13 holothurian species of the orders Elasipodida, Holothuriida, and Synallactida, from bulk stable isotope analyses (δ13C, δ15N) of holothurian body walls and guts, gut contents, and feces that were combined with compound-specific stable isotope analyses of amino acids, phospholipid-derived fatty acids, and neutral lipid-derived fatty acids in the body wall. Fatty acid concentrations showed high levels of storage lipids, an likely adaption to limited food supply to abyssal plains. Amino acid δ15N isotope values allowed estimating trophic levels of holothurian species and calculating heterotrophic re-synthesis of amino acids. Fatty acids served as trophic markers for feeding on diatom- and dinoflagellate derived phytodetritus, bacteria, Foraminifera, and detritus containing the PUFA C22:1ω9-cis. Several holothurian species seemed to be secondary consumers of detritus, while bacteria in their guts were primary consumers of this detritus. A Sorensen-Dice coefficient based cluster analysis using data of trophic levels, levels of heterotrophic re-synthesis of amino acids, feeding selectivity, and food sources/ diet suggested three trophic groups, characterized by different trophic levels. We show that this multi-biomarker driven approach allows to deconvolve trophic niches and feeding selectivity in one of the most challenging environments on earth and to identify dependence of deep-sea species to organic matter inputs that vary with season and/or climate.