Devils Hole, a unique ecosystem in the Mojave Desert, is home to a few dominant species of algae and invertebrates as well as the endangered Devils Hole pupfish, Cyprinodon diabolis. With consistently high water temperature (33.5 °C/93 °F) and low dissolved oxygen (O₂) concentration (about 2.5 mg O₂ · L^-1), organisms are at the extremes of their physiological limits, and production of O₂ by microbial biofilms is essential to ecosystem stability. Water column O₂ concentrations were measured from July 2008 to March 2010 in the deep pool and shallow shelf habitats of Devils Hole to quantify variability in O₂ concentrations and ecosystem metabolism rates. Benthic O₂ dynamics were also measured in microbial biofilms using microelectrode surveys. Water column O₂ ranged from 2 to 6 mg O₂ · L^-1 in summers and from 1.5 to 2.2 mg O₂ — L^-1 in winter across the deep pool and shallow shelf habitats. Primary production ranged from 4 to 21 mg O₂ · L^-1d^-1, with a significant decline over the study period, potentially due to a change in the microbial biofilm community. Respiration ranged from 1.5 to 9.7 mg O₂ · L^-1d^-1 and showed a significant increase over time. Within microbial biofilms, O₂ ranged from 0 to 76 mg O₂ * L^-1. Higher concentrations of O₂ produced by these microbial biofilms may be due to improved photosynthetic efficiency under limited sunlight exposure. Autotrophic biofilms had higher O₂ concentrations during direct light exposure than during indirect light exposure. In contrast, heterotrophic biofilms had similar O₂ concentrations regardless of light exposure. Because microbial biofilms are important components of this unique ecosystem, shifts in their composition or activity may threaten ecosystem stability by reducing background O₂ concentrations below the physiological limits of the endangered Devils Hole pupfish and the entire biotic community.