We characterized the biotic integrity of the sub‐tropical Duero River, Mexico, comparing current and past index of biotic integrity (IBI) scores with results of an environmental quality index (EQI), inventorying abiotic characteristics and human impacts, and documenting ecosystem changes over the last three decades. We sampled the fish community and measured environmental variables during the dry season of 2009 at 19 sites. We compared 2009 findings with fish data obtained in 1986 and 1991 at nine of the sites. The correlation between the IBI and EQI was high (r2 = 0.79, p = 0.0002), indicating that the IBI accurately characterized environmental condition. Thirteen sites were degraded (68%) based on IBI scores, three were fair (16%) and three had good conditions (16%). Based on the EQI, 12 sites were classified as poor (63%), two as fair (11%) and five as good (19%). A cluster analysis of nine abiotic variables and the IBI values yielded three groups: six sites with good integrity and environmental quality, eight with high chemical oxygen demand and four with high total dissolved solids. A discriminant analysis (square canonical correlation: 0.922, and Wilks' lambda significance: 0.001) identified biochemical oxygen demand, distance to vegetation and total dissolved solids as the main predictors. Compared to 1986 and 1991, 2009 IBI values and ratings had declined significantly (multiple response permutation procedure A = 0.16, p = 0.0005), indicating a trend of environmental deterioration, with scores and ratings at 7 of 9 sites lower in 2009. One site had declined from good to fair, one from good to poor, five from fair to poor, one had remained fair and one had remained good. Degradation was related to excessive human water use, reduced river flows, increased wastewater discharge, deforestation, erosion, and invasive species, and mismanagement of preserved sites (springs), all of which highlight an urgent need to reverse declining environmental conditions. Copyright © 2014 John Wiley & Sons, Ltd.