Gut content analysis using molecular techniques can help elucidate predator-prey relationships in situations in which other methodologies are not feasible, such as in the case of trophic interactions between minute species such as mites. We designed species-specific primers for a mite community occurring in Spanish citrus orchards comprising two herbivores, the Tetranychidae Tetranychus urticae and Panonychus citri, and six predatory mites belonging to the Phytoseiidae family; these predatory mites are considered to be these herbivores’ main biological control agents. These primers were successfully multiplexed in a single PCR to test the range of predators feeding on each of the two prey species. We estimated prey DNA detectability success over time (DS50), which depended on the predator-prey combination and ranged from 0.2 to 18 h. These values were further used to weight prey detection in field samples to disentangle the predatory role played by the most abundant predators (i.e. Euseius stipulatus and Phytoseiulus persimilis). The corrected predation value for E. stipulatus was significantly higher than for P. persimilis. However, because this 1.5-fold difference was less than that observed regarding their sevenfold difference in abundance, we conclude that P. persimilis is the most effective predator in the system; it preyed on tetranychids almost five times more frequently than E. stipulatus did. The present results demonstrate that molecular tools are appropriate to unravel predator-prey interactions in tiny species such as mites, which include important agricultural pests and their predators. We are especially grateful to Beatriz Sabater (Trinity College of Dublin), Ernestina Aguilar-Fenollosa, Loredana Scalschi and Amelia Simó (UJI) for technical advice, to Begonya Vicedo (UJI) for logistical support and Juan Ramón Boyero (IFAPA-Churriana, Málaga), Pablo Alvarado (Laboratorio de Producción y Sanidad Vegetal, Huelva), Ahlem Harbi, Khaled Abbes and Poliane Sá Argolo (IVIA) for their help in mite sampling. Furthermore, we want to express our gratitude to four anonymous reviewers for their helpful comments on the manuscript. This work was partially funded by the Spanish Ministry of Science and Innovation (Projects: AGL2008-05287-C04/AGR and AGL2011-30538-C03-01) and the Bancaixa Foundation - Universitat Jaume I Research Program (P1-1A2005-03 and P11B2008-02). T. Pina was the recipient of a postdoctoral grant (PICD) from UJI. J. A. Jaques is the head of the Integrated Pest Management research group at UJI and was involved in the design and discussion of the assays. M. A. Hurtado led the molecular biology approach used in this study and designed all the experiments except the qPCR, which was developed by G. Camañes. C. Pérez-Sayas and T. Pina performed all the experiments with support from M. A. Gómez-Martínez. M. V. Ibáñez-Gual and T. Pina statistically analysed the results, which were discussed by all authors. All authors contributed to writing of the manuscript.