Ecological understanding of the role of consumer-resource interactions in natural food webs is limited by the difficulty of accurately and efficiently determining the complex variety of food types animals have eaten in the field. We developed a method based on DNA metabarcoding multiplexing and next-generation sequencing to uncover different taxonomic groups of organisms from complex diet samples. We validated this approach on 91 faeces of a large omnivorous mammal, the brown bear, using DNA metabarcoding markers targeting the plant, vertebrate, and invertebrate components of the diet. We included internal controls in the experiments and performed PCR replication for accuracy validation in post-sequencing data analysis. Using our multiplexing strategy, we significantly simplified the experimental procedure and accurately and concurrently identified different prey DNA corresponding to the targeted taxonomic groups, with ≥60% of taxa of all diet components identified to genus/species level. The systematic application of internal controls and replication was a useful and simple way to evaluate the performance of our experimental procedure, standardize the selection of sequence filtering parameters for each marker data, and validate the accuracy of the results. Our general approach can be adapted to the analysis of dietary samples of various predator species in different ecosystems, for a number of conservation and ecological applications entailing large-scale population level diet assessment through cost effective screening of multiple DNA metabarcodes, and the detection of fine dietary variation among samples or individuals and of rare food items. This article is protected by copyright. All rights reserved.