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Abstract Reduced belowground carbon (C) allocation by plants exposed to ozone may change properties and activities of the microbial community in soils. To investigate how soil microbial biomass and extracellular enzyme activities respond to elevated ozone, we collected soils from a temperate grassland after four years of ozone exposure under fully open-air field conditions. We measured soil microbial biomass, the metabolism of low molecular weight C substrates and hydrolytic extracellular enzyme activities in both bulk soil and isolated aggregates to assess changes in microbial activity and community function. After four years of elevated ozone treatment, soil total organic C was reduced by an average of 20% compared with ambient condition. Elevated ozone resulted in a small but insignificant reduction (4–10%) in microbial biomass in both bulk soil and isolated aggregates. Activities of extracellular enzymes were generally not affected by elevated ozone, except β -glucosidase, whose activity in bulk soil was significantly lower under elevated ozone than ambient condition. Activities of β -glucosidase, leucine aminopeptidase and acid phosphatase were higher in microaggregates (<0.25 mm) as compared to macroaggregates (>0.25 mm). Elevated ozone had no effects on mineralization rates of low molecular weight C substrates in both bulk soil and isolated aggregates. We therefore conclude that the size and activity rather than function of the soil microbial community in this semi-natural grassland are altered by elevated ozone. Graphical abstract Unlabelled Image Highlights • A temperate grassland was exposed to ozone for 4 years under field conditions. • Soil total carbon and β-glucosidase activity were decreased under elevated ozone. • Neither ozone nor its interaction with aggregate size affected microbial activities. • Elevated ozone could alter the size and activity of soil microbial community. [ABSTRACT FROM AUTHOR]