Abstract We studied the effects of O 3 and CO 2 alone and in combination on soil microbial communities by assessing the changes in total PLFA biomass, profiles and specific subgroups. Meadow mesocosms were exposed to slightly elevated O 3 (40–50 ppb) and CO 2 (+100 ppm) in open-top chambers for three subsequent growing seasons (2002–2004). Decreased total, bacterial, actinobacterial, fungal PLFA biomass values as well as fungal:bacterial PLFA biomass ratio were measured after three growing seasons of fumigations with elevated O 3 . There were significant differences in the relative proportions of individual PLFAs between the control and elevated O 3 treatments. Moreover, enhanced O 3 alone and in combination with CO 2 modified the structure of the microbial community. The effects of elevated CO 2 given alone on PLFA profiles were negligible. Our results show that elevated O 3 alone and in combination with CO 2 even at moderate levels may cause changes in the biomass and composition of the microbial community in meadow soils, which may lead to functional changes in soil ecosystem processes.