Cold seep communities in the Mediterranean Sea have only been discovered two decades ago, and their trophic ecology has been the subject of very few studies. We investigated the benthic food web of two deep chemosynthesis-based ecosystems on the Napoli and Amsterdam mud volcanoes (MVs) in the eastern Mediterranean Sea (~2,000 m depth). Seeping methane has been detected at the surface of both MVs during pioneering cruises and has been hypothesised to be assimilated by benthic fauna as observed in other oceans' margins. Given the extreme oligotrophic character of the eastern Mediterranean Sea, we a priori expected that chemosynthetic food sources, especially methane-derived carbon (MDC), played a major trophic role in these deep seep communities relative to what has been observed in other seep systems worldwide. We aimed at unravelling the trophic relationships on Napoli and Amsterdam MVs through the analysis of carbon, nitrogen and sulphur isotopes both in the dominant benthic invertebrates including the small endofauna (300 μm < size < 1 cm) and in the sedimented organic matter. In particular, we assessed the fraction of MDC in the tissue of several heterotrophic and symbiotic species. Low mean δ34S and δ13C values (0.4 ± 4.8‰ and −31.6 ± 5.7‰, respectively) obtained for mega- and macrofauna suggested that the investigated benthic food webs are virtually exclusively fuelled by carbon of chemosynthetic origin. A few grazer invertebrates (δ34S up to 11‰) depart from this trend and could complement their diet with sedimented and decayed phytoplanktonic organic matter. Faunal δ13C values indicated that the oxidation of sulphur is likely the predominant energetic pathway for biosynthesis on both MVs. Nevertheless, mytilid bivalves and small capitellid, ampharetid and spionid polychaetes were 13C-depleted (δ13C < −37‰) in a way indicating they assimilated a significant portion of MDC. For these later heterotrophic species, MDC ranged between 21 and 31% (lower estimates) and 97 and 100% (upper estimates). However, our results highlighted that the origin of assimilated carbon may be complex for some symbiotic species. The vestimentiferan tubeworm Lamellibrachia sp., which exclusively depends on its sulphur-oxidising endosymbionts, showed a ~20‰ inter-individual δ13C variability on a very small spatial scale (<1 m) at the summit of Napoli MV. This mostly reflects the variable isotopic composition of pore-water-dissolved inorganic carbon (DIC) and evidenced that tubeworms (and subsequently their endosymbionts) uptake DIC derived from multiple methane oxidation processes in varying proportions. The lower and upper MDC estimates for the vestimentum of Napoli's individuals were 11-38 and 21-73%, respectively. Finally, data on trophic ecology of Napoli and Amsterdam MVs clearly corroborate previous geophysical results evidencing the spatial heterogeneity of Mediterranean MV environmental conditions.