Difloxacin (DIF) belongs to the fluoroquinolones, a frequently detected group of antibiotics in the environment. It is excreted in pig manure to a large extent and may consequently reach soils in potentially effective concentrations via manuring. The aim of this study was to assess the effects of DIF-spiked manure on microbial communities and selected functions in soils in a microcosm experiment up to 1 month after application. To test a dose dependency of the effects, three different concentrations of DIF (1, 10 and 100 mg/kg of soil) were used. Microcosms with application of pure manure, as well as untreated microcosms served as control. The addition of pure manure resulted in an increase of microbial biomass and soil respiration as well as a reduced bacteria/fungi ratio. Due to the fast and strong immobilisation of DIF, effects of the antbiotic compound were only visible up to 8 days after application (microbial biomass; respiration; potential denitrification; ratio of bacteria/fungi). As expected these short-term effects resulted in reduced potential denitrification rates as well as a reduced bacteria/fungal ratio in the treatments were DIF has been applied. Surprisingly, microbial biomass values as well as respiration rates were increased by DIF application. Other parameters like nitrate and ammonium content in soil were not influenced by DIF application at any time point. Long-term effects (32 days after application) were only visible for the potential nitrification rates. For those parameters that were influenced by the DIF application a clear dose dependency could not be described.