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Natural floodplains comprise a spatial mosaic of habitat types that vary in successional development. The physical-chemical properties of soils and sediments in these habitats vary spatio-temporally with changes in hydrology, thereby influencing microbial dynamics and general floodplain functioning. The major goals of this study were to characterize the spatio-temporal variation of different floodplain habitats and elucidate microbial function as a mechanistic basis underlying floodplain functioning. Microbial functional parameters (i.e. soil-sediment respiration, enzyme activity) as well as a microbial state variable (i.e. bacterial abundance, BA) were assessed in conjunction with abiotic properties of respective soils and sediments in different floodplain habitats over an annual cycle. BA and respiration differed significantly among habitat types, being lowest in early successional gravel and stream channel habitats and highest in older successional habitats of mature forest, islands, and riparian pasture/grassland. Coarse-scale floodplain hydrology influenced the spatial variation in microbial measures within specific habitats. A non-metric multidimensional scaling clearly separated habitat types based on the functional activities of eight tested enzymes and specific microbial variables could be linked to soil-sediment respiration. These results document a high functional heterogeneity of soil and sediment microbial variables that complements the shifting habitat mosaic found in most natural floodplains. Moreover, the use of functional measures such as respiration enhances the understanding of complex floodplain functioning.