Sub-seafloor hydrothermal processes along volcanically active plate boundaries are integral to the formation of seafloor massive sulfide deposits and to oceanic iron cycling, yet the nature of their relationship is poorly understood. Here we apply iron isotope analysis to sulfide minerals from the Trans-Atlantic Geotraverse (TAG) mound and underlying stockwork, 26°N Mid-Atlantic Ridge, to trace hydrothermal processes inside an actively-forming sulfide deposit in a sediment-free mid-ocean ridge setting. We show that data for recently formed chalcopyrite imply hydrothermal fluid–mound interactions cause small negative shifts (<−0.1‰) to the δ56Fe signature of dissolved iron released from TAG into the North Atlantic Ocean. Texturally distinct types of pyrite, in turn, preserve a δ56Fe range from −1.27 to +0.56‰ that reflects contrasting precipitation mechanisms (hydrothermal fluid–seawater mixing vs. conductive cooling) and variable degrees of progressive hydrothermal maturation during the >20 kyr evolution of the TAG complex. The identified processes may explain iron isotope variations found in fossil onshore sulfide deposits.