Multiple S isotope systematics (δ34S and Δ33S) and high resolution in-situ S isotope measurements offer new perspectives on the study of biological and abiotic cycling of sulfur in hydrothermal systems. We applied these techniques to the Tethyian Troodos ophiolite (91 Ma) from Cyprus, one of the best-preserved remnants of oceanic crust in the world, using materials from deep drill cores and surface sampling. We focused on the volcanic section of the ophiolite, including the hydrothermal massive sulfide deposit at Agrokipia, which represents a fossil zone of high-temperature fluid upwelling, and the Akaki river section which displays a range of lower temperature alteration types.

The δ34S and Δ33S values of bulk and SIMS (secondary ion mass spectrometry) analyses from the Agrokipia sulfide deposits show that the sulfide minerals are largely derived from thermochemical reduction of entrained seawater sulfate and leached H2S from the “root zone” of hydrothermal upwelling. The contributions of these two sources can vary substantially within individual sulfide grains, indicating a very dynamic mixing between these sulfur sources. Microbial reworking of the sulfide mound is recorded in a sample with very elevated Δ33S values (0.22‰).

The Akaki and Agrokipia volcanics experienced low temperature sulfur loss and removal of heavier sulfur isotopes due to partial oxidation by microbes. While some intervals gained sulfur, and have δ34S and Δ33S values indicative of microbial sulfate reduction. REE-data of vein quartz containing pyrite with δ34S = ~− 21‰ implies local ephemeral hydrothermal upwelling in the lower Akaki volcanics, possibly associated with the late stage boninitic magmatic activity in the Troodos ophiolite, suggesting that microbial sulfate reduction in oceanic crust may continue for 10–15 Ma in crustal sections with prolonged igneous activity such as Troodos.