Our objective in this study was to characterize prokaryotic sulphide production within the oxygenic, predominantly eukaryotic algal mat in an acidic stream, Nymph Creek, in Yellowstone National Park (YNP). We used microsensors to examine fluctuations in H2S and O2 concentrations over time through the vertical aspect of the ~3 mm mat in a 46–48°C region of the creek. We also used analyses of PCR-amplified 16S rRNA gene sequences obtained from denaturing gradient gels, and PCR-amplified sequences of a functional gene associated with microbial sulphate respiration (dsrA) to characterize the bacterial community in the same region of the mat. During midday, photosynthesis rates were high within the first 500um interval of the mat and high oxygen concentrations (600% air saturation) penetrated deeply (>1800 um) into the mat. During early evening and night, oxygen concentrations within the first 1100 um of the mat decreased over time from 60% air saturation (a.s) to 12% a.s. A precipitous decline in oxygen concentration occurred at a depth of 1100 um in all night measurements and anoxic conditions were present below 1200 um. Within this anoxic region, sulphide concentrations increased from nearly 0 uM at 1200 um depth to 100 uM at 2400 um depth. Enrichment cultures inoculated with Nymph Creek mat organisms also produced H2S. Sequence analyses of 16S rRNA and dsrA genes indicated the presence of at least five bacterial genera including species involved in dissimilative sulphate or sulphur reduction.