Unraveling the potentially shifting controls over microbial activity among habitats and across seasonal transitions is critical for understanding how freshwater ecosystems influence broader elemental cycles, and how these systems may respond to global changes. We used nutrient-diffusing substrates to investigate seasonal patterns and constraints on microbial activity of biofilms in streams draining distinct landscape features of the boreal biome (forests, mires, and lakes). Microbial respiration (MR) largely mirrored spatial and temporal variation in water temperature. However, limitation by labile carbon (C) was a constraint to microbial activity during ice-covered periods, when MR of control nutrient-diffusing substrates fell below rates predicted from stream temperature alone. Variation in C limitation among the study streams was reflective of putative organic C availability, with C limitation of biofilms weakest in the dissolved organic C (DOC)-rich, mire-outlet stream and greatest in the relatively DOC-poor, forest stream. Incidences of nutrient limitation were only observed during warmer months. Our study illustrates how variation in processes mediated by heterotrophic biofilms and seasonal shifts in resource limitation can emerge in a stream network draining a heterogeneous landscape. In addition, our results show that, for a large portion of the year, heterotrophic processes in boreal streams can be strongly limited by the availability of labile C, despite high DOC concentrations. Metabolic constraints to dissolved organic matter processing at near-freezing temperatures, coupled with hydrological controls over the delivery of more labile organic resources to streams (e.g., soil freezing and flooding), have potentially strong influences on the productivity of boreal streams.