Mayfly (Hexagenia spp.) nymphs have been widely used as indicators of water and substrate quality in lakes. Thermal stratification and the subsequent formation of benthic hypoxia may result in nymph mortality. Our goal was to identify potential associations between recent increases in temperature and eutrophication, which exacerbate hypoxic events in lakes, and mayfly populations in Lake Erie. Nymphs were collected during April–May 1999–2014. We used wind and temperature data to calculate four measures of thermal stratification, which drives hypoxic events, during summers of 1998–2013. Bottom trawl data collected during August 1998–2013 were used to estimate annual biomass of fishes known to be predators of mayfly nymphs. We used Akaike's Information Criterion to identify the best one- and two-predictor regression models of annual population densities (N/m²) of age-1 and age-2 nymphs, in which candidate predictors included the four measures of stratification, predator fish biomass, competition, and population densities of age-2 (for age-1) and age-1 (for age-2) nymphs from the previous year. Densities of both age classes of nymphs declined over the time series. Population densities of age-1 and age-2 nymphs from the previous year best predicted annual population densities of nymphs of both age classes. However, hypoxic conditions (indicated by stratification) and predation both had negative effects on annual population density of mayflies. Compared with predation, hypoxia had an inconsistent effect on annual nymph density. The increases in temperature and eutrophication in Lake Erie, which exacerbate hypoxic events, may have drastic effects on the mayfly populations.