Microorganisms are key drivers of leaf litter decomposition; however, the mechanisms underlying the dynamics of different microbial groups are poorly understood. We investigated the effects of seasonal variation and invertebrates on fungal and bacterial dynamics, and on leaf litter decomposition. We followed the decomposition of Populus nigra litter in a Mediterranean stream through an annual cycle, using fine and coarse mesh bags. Irrespective of the season, microbial decomposition followed two stages. Initially, bacterial contribution to total microbial biomass was higher compared to later stages, and it was related to disaccharide and lignin degradation; in a later stage, bacteria were less important and were associated with hemicellulose and cellulose degradation, while fungi were related to lignin decomposition. The relevance of microbial groups in decomposition differed among seasons: fungi were more important in spring, whereas in summer, water quality changes seemed to favour bacteria and slowed down lignin and hemicellulose degradation. Invertebrates influenced litter-associated microbial assemblages (especially bacteria), stimulated enzyme efficiencies and reduced fungal biomass. We conclude that bacterial and fungal assemblages play distinctive roles in microbial decomposition and differ in their sensitivity to environmental changes, ultimately affecting litter decomposition, which might be particularly relevant in highly seasonal ecosystems, such as intermittent streams.