Aquatic bryophytes commonly form mats in small, headwater streams that trap sediment and nutrients and provide substrate for epiphytic algae. However, aquatic bryophytes are rarely included in our current conceptual models of stream ecosystem function or biogeochemical cycling. Here, we examine the potential for the “bryosphere” - inclusive of live and senescent bryophyte tissue, trapped particulates, and associated organisms - to contribute to in-stream biogeochemical function. We measured nitrogen (N) fixation rates from samples of two species of aquatic bryophytes - a liverwort (Marsupella emarginata) and a moss (Codriophorus aduncoides) - collected from Hubbard Brook Experimental Forest (New Hampshire, USA) and examined how N fixation rates vary by horizon (live/senescent tissue) and mat thickness. All incubated samples demonstrated measurable N fixation (mean ± s.d. = 70 ± 26 µg N m-2 hr-1). We found scant evidence of epiphytic cyanobacteria, but nearly all samples had δ15N signatures indicative of N fixation (1 to -3‰), suggesting potential contributions from endophytic communities. When comparing between live and senescent tissues, N fixation was greater in the live, green horizon, and in samples collected from shallower mats (<5 cm thick), likely due to shallow mats consisting primarily of live tissues. The streams from which these aquatic bryophytes were collected are very oligotrophic, but our findings suggest aquatic bryophytes are nevertheless able to access a “new” source of nitrogen. We propose that estimates of bryophyte coverage and nitrogen fixation should be included in stream N budgets, even when stream water nutrient concentrations remain low.