Newly discovered hydrothermal systems in the Pescadero Basin (PB) and the neighboring Pescadero Transform Fault (PTF) at the mouth of the Gulf of California disclosed a diverse macrofauna assemblage. The trophic structure of both ecosystems was assessed using carbon (δ13C), nitrogen (δ15N), and sulfur (δ34S) stable isotopes. The δ13C ranged from -40.8 to -12.1‰, revealing diverse carbon sources and its assimilation via Calvin-Benson-Bassham and the reductive tricarboxylic acid cycles. The δ15N values were between -12.5 and 18.3‰, corresponding to primary and secondary consumers. The δ34S values fluctuated from -36.2 to 15.1‰, indicating the sulfide assimilation of biogenic, magmatic, and photosynthetic sources. In PB high-temperature vents, primary consumers including symbiont-bearing, bacterivores and filter-feeders predominated. The secondary consumers within the scavengers/detritivores and predator guilds were scarce. The siboglinid Oasisia aff alvinae dominated the macrofauna assemblage at PB, but rather than playing a trophic role, it provides a substrate to vent dwellers. In PTF low-temperature vents, only symbiont-bearing primary consumers were analyzed, displaying the lowest δ34S values. This assemblage was dominated by the coexisting siboglinids Lamellibrachia barhami and Escarpia spicata. δ34S values allowed to distinguish between PB and PTF vent communities, to exclude the presence of methanotrophic organisms, and the detection of photosynthetic organic matter input.