Environmental variability in space and time is a primary mechanism allowing species that share resources to coexist. Fluctuating conditions are a double edged sword for diversity, either promoting coexistence through temporal niche partitioning or excluding species by stochastic extinctions. The net effect of environmental variation on diversity is largely unknown. We examined the association between zooplankton species richness in lakes and environmental variability on interannual, seasonal and shorter time scales, as well as long-term average conditions. We analyzed data on physical, chemical and biological limnology in 53 temperate zone lakes in North America and Europe sampled over a combined 1042 years. Large fluctuations in pH, phosphorus and dissolved organic carbon concentration on different time scales were associated with reduced zooplankton species richness. More species were found in lakes that showed greater temperature variation on all time scales. Environmental variability on different time scales showed similar or, in some cases, stronger associations with zooplankton species richness compared with long-term average conditions. Our results suggest that temporal fluctuations in the chemical environment tend to exclude zooplankton species while temperature variability promotes greater richness. The results indicate that anthropogenic increases in temporal variability of future climates may have profound effects on biodiversity.