Metazoan consumption of Bacteria and Archaea may impact key microbially mediated processes, yet techniques to quantify trophic linkages require laboratory evaluation before robust application to field observations are possible. In this laboratory-based study, an annelid belonging to a genus common at reducing habitats, Ophryotrocha labronica, was raised on eukaryotic, bacterial or archaeal food sources to test whether fatty acid and stable isotope paradigms are appropriate to quantitatively model metazoan consumption of microbial food sources. This annelid's δ13C tissue-diet shift, −3.6‰ to +3.6‰, was neither zero nor constant and was a function of the δ13C value and the C : N ratio of its food. Nitrogen isotope (mean δ15N) tissue-diet shifts ranged from −0.8‰ to −3.0‰ when raised in antibiotic-laden seawater in contrast to the tissue-diet shifts of +2.3 to +3.4 that are often used to define a species’ trophic level. The use of antibiotics to control microbial growth during the experiment appeared to have little effect on these fractionation values, except for individuals fed Spinacia oleracea that may have been impacted by the antibiotic treatment; when antibiotics were not used the δ15N value of O. labronica was not different from the S. oleracea that it consumed. Fatty acid profiles within this species were largely independent of diet. Polyunsaturated fatty acids, commonly used as a metric for phytoplankton consumption and not provided by the food sources, were present in O. labronica tissues regardless of food source. This suggests that O. labronica can synthesize these fatty acids. Whereas field studies have shown the importance of biomarker approaches, tissue-diet shifts based on the literature could erroneously characterize the diet and trophic level of this species. This is especially true in food webs that are known to rely on microbial production, such as those found at deep-sea chemosynthetic habitats.