AbstractIncreasing plant species richness has been shown to positively affect the diversity of a range of other organisms, both above‐ and belowground. Although ants have a multitude of interactions with other organisms through their role as predators and mutualists, ants are a taxon that has rarely been investigated in studies of biodiversity effects. Ants are known to respond to changes in microclimatic conditions such as temperature and humidity, and these microclimatic conditions are known to be affected by vegetation characteristics such as standing biomass, which in turn can be affected by plant diversity. We investigated the effects of plant species richness (1–60), the number of plant functional groups (FGs; 1–4), and the presence of particular FGs on the occurrence and activity of ants, in the context of a grassland biodiversity experiment. Ant abundance, estimated as the mean number of workers in pitfall traps, and the number of colonies within plots, but not ant species richness, were negatively affected by plant species richness, and also by the identity of plant FGs, particularly legumes (negative effects) and grasses (positive effects). Statistical approaches showed that these effects were largely mediated by biotic (aboveground plant biomass: negative effect) and abiotic variables (soil temperature: positive effect). Notably, ant activity, measured as the mean number of workers foraging on baits and with a disproportionate dominance of a single species (Lasius niger), showed a more complex pattern, where plant species richness and number of FGs interacted to explain differential attractiveness to different resources (represented by sugar and tuna baits), likely representing current nutritional requirements for ants. Similarly, increasing soil temperature increased the level of ant activity, indicating that abiotic factors might impose thermal constraints on ectotherms inhabiting temperate grasslands. Our results indicate that a higher number of plant species and higher plant biomass had a negative effect on ants in experimental grasslands. Understanding the responses of an ecologically important group such as ants will allow us to infer the effects that biodiversity loss could have on animal‐mediated ecosystem processes.