Filamentous large sulfur-oxidizing bacteria (FLSB) of the family Beggiatoaceae are globally distributed aquatic bacteria that can control geochemical fluxes from the sediment to the water column through their metabolic activity. FLSB mats from hydrothermal sediments of Guaymas Basin, Mexico, typically have a “fried-egg” appearance, with orange filaments dominating near the center and wider white filaments at the periphery, likely reflecting areas of higher and lower sulfide fluxes, respectively. These FLSB store large quantities of intracellular nitrate that they use to oxidize sulfide. By applying a combination of 15N-labeling techniques and genome sequence analysis, we demonstrate that the white FLSB filaments were capable of reducing their intracellular nitrate stores to both nitrogen gas and ammonium by denitrification and dissimilatory nitrate reduction to ammonium (DNRA), respectively. On the other hand, our combined results show that the orange filaments were primarily capable of DNRA. Microsensor profiles through a laboratory-incubated white FLSB mat revealed a 2- to 3-mm vertical separation between the oxic and sulfidic zones. Denitrification was most intense just below the oxic zone, as shown by the production of nitrous oxide following exposure to acetylene, which blocks nitrous oxide reduction to nitrogen gas. Below this zone, a local pH maximum coincided with sulfide oxidation, consistent with nitrate reduction by DNRA. The balance between internally and externally available electron acceptors (nitrate) and electron donors (reduced sulfur) likely controlled the end product of nitrate reduction both between orange and white FLSB mats and between different spatial and geochemical niches within the white FLSB mat.