Native grasslands have been vastly transformed with the expansion of human activities. Applied fire regimes offer conservation-based management an opportunity to enhance remaining grassland biodiversity and secure its persistence into the future. Fire regimes have complex interactions with abiotic and biotic ecosystem components that influence environmental heterogeneity and biodiversity. We examined the pyrodiversity–biodiversity hypothesis, which suggests that more species are supported where pyrodiversity, that is, the level of environmental heterogeneity associated with different fire regimes, is greater. A mesocosm-type field experiment, maintained for 38 yr, was used to determine the response of plant diversity to 1-, 2-, 5- and 12-yr fire-return interval treatments, with early-dormant, middormant and early–growing season burns. Our sampling regime was designed to assess the influence of fire treatments and combinations thereof, over spatial scale, on plant diversity. Pyrodiversity was maximized where fire regime diversity, simulated by varying the size of patches with different fire treatments, was greatest. Species richness was predicted to be reduced at short and long extremes of fire-return interval, as suggested by the intermediate-disturbance hypothesis. The influence of fire treatments on alpha and beta diversity, and plant functional groups, were tested using multivariate and Bayesian models. Multilevel models of plant height and growth form, with fire-return interval, reflected the strong indirect influence of fire-return interval on sward structure and the plant environment. The pyrodiversity–biodiversity and intermediate-disturbance hypotheses were only partially supported and depended on the plant group and spatial scale of assessment. Although both frequent and infrequent burns made important contributions to overall species richness, richness peaked where 20–40% of the area was protected from frequent fires. The larger contribution of frequent burning to diversity was due to an interaction with scale and forb turnover over the trial area. Extremes in fire-return intervals reduced forb richness, supporting the predictions of the intermediate-disturbance hypothesis. Spring burns had a weak negative influence on forb alpha diversity, but only at small scales. For a meaningful contribution of management to plant diversity, traditional fixed biennial burns need to be supplemented with smaller patches burned with longer fire-return intervals, and extremes in fire-return intervals avoided.