Knowledge on microbial community shifts during ecosystem succession from bare surfaces resulting from massive landscape stripping is extremely limited. Here we took advantage of an artificially created experimental catchment (6 ha) to assess structural and functional changes of microbial communities in ephemeral stream sediments and adjacent soils between 3 and 13 years after catchment construction. The catchment has since developed in undisturbed conditions, with major transformations in its morphology, hydrology and vegetation reflected by changes in microbial community structure and function. Initially dominated by cyanobacteria (42% of 16S rRNA reads in 2008 and 0.3% in 2018), the bacterial community shifted to an essentially heterotrophic composition within 10 years, when Alphaproteobacteria (12 vs 21%) and Planctomycetes (3 vs 16%), in particular, gained in importance. Similarly, Sordariomycetes (5% of ITS reads in 2008 and 27% in 2018) replaced Dothideomycetes (53 vs 14%) as the prevailing fungal class. Microbial respiration rates increased tenfold, from an average of 0.5–4.4 μg CO2 g−1 DM h−1, accompanied by an increase in potential enzyme activities. Seasonal patterns of microbial community functions were accentuated over a decade of catchment development, whereas structural community changes were less pronounced. Spatial variation of community composition also increased, with differences between soils and sediments intensifying over time. However, a striking disconnect between microbial community structure and function in 2008 had vanished by 2018. Thus, a decade of ecosystem succession resulted in fundamental shifts in microbial community structure and function, highlighting the intricate interplay between changing environmental conditions and microbial responses.