Abstract A novel combination of the technique of diffusive gradients in thin films (DGT) and laser ablation high-resolution multicollector inductively coupled plasma mass spectrometry was developed to study sulphur isotope variations of dissolved pore-water sulphide in freshwater and marine sediments. The technique enables two-dimensional mapping of isotopic variations (δ 34S) in dissolved sulphide captured as solid Ag 2S in DGT polyacrylamide gels. Measurements can be performed at a spatial resolution (~ 100 μm) relevant to microbiological processes and formation of individual iron sulphide grains in surface sediments. Values of δ 34S measured in BaSO 4–DGT gel isotope standards (δ 34S = 9.28 ± 0.36‰ to 9.33 ± 0.57‰) are within 1‰ of the accepted value determined with conventional analytical techniques (δ 34S = 10.13 ± 0.29‰). Sulphur isotope measurements were performed in sediments from a eutrophic lake (Esthwaite Water, UK) contained in laboratory mesocosms. Bacterial sulphate reduction and sulphide formation in this sediment are predominantly localized to discrete, mm-sized microniches, where oxidation of labile organic matter such as fresh algae and faecal pellets drives the reduction of sulphate. The results emphasize the importance of microniches as localized, highly dynamic reaction sites in sediments, where significant shifts in δ 34S of up to + 20‰ relative to the local background were measured across microniches. The improved spatial resolution for pore-water sulphur isotope measurements, compared to that of conventional sampling and analytical techniques, is essential for improving our understanding of the global biogeochemical cycling of sulphur as well as trace metal–sulphide interactions in modern sediments.