Karst aquifers and springs provide the dissolved oxygen critical for survival of endemic stygophiles worldwide, but little is known about fluctuations of dissolved oxygen concentrations (DO) and factors that control those concentrations. We investigated temporal variation in DO at Barton Springs, Austin, Texas, USA. During 2006–2012, DO fluctuated by as much as a factor of 2, and at some periods decreased to concentrations that adversely affect the Barton Springs salamander (Eurycea sorosum) (≤4.4 mg/L), a federally listed endangered species endemic to Barton Springs. DO was lowest (≤4.4 mg/L) when discharge was low (≤1 m3/s) and spring water temperature was >21 °C, although not at a maximum; the minimum DO recorded was 4.0 mg/L. Relatively low DO (<6 mg/L) also was measured at relatively high discharge (3.2 m3/s) and maximum T (22.2 °C). A four-segment linear regression model with daily data for discharge and spring water temperature as explanatory variables provided an excellent fit for mean daily DO (Nash–Sutcliffe coefficient for the validation period of 0.90). DO also fluctuated at short-term timescales in response to storms, and DO measured at 15-min intervals could be simulated with a combination of discharge, spring temperature, and specific conductance as explanatory variables. On the basis of the daily-data regression model, we hypothesize that more frequent low DO corresponding to salamander mortality could result from (i) lower discharge from Barton Springs resulting from increased groundwater withdrawals or decreased recharge as a result of climate change, and (or) (ii) higher groundwater temperature as a result of climate change.