1. Microbial activity plays a central role in nitrogen (N) cycling, with effects on forest productivity. Though N bio-transformations, such as nitrification, are known to occur in the soil, here we investigate whether nitrifiers are present in tree canopies and actively process atmospheric N. 2. This study was conducted in a Mediterranean holm oak (Quercus ilex L.) forest in Spain during the transition from hot dry summer to cool wet winter. We quantified NH4+—N and NO3-—N fluxes for rainfall (RF) and throughfall (TF) and used δ15N, δ18O, and Δ17O to elucidate sources of NO3-. Finally, we characterized microbial communities and abundance of nitrifiers on foliage, RF and TF water through metabarcoding and quantitative Polymerase Chain Reaction, respectively. 3. NO3—N fluxes at the site were larger in TF than RF, suggesting a contribution from dry deposition, as also supported by δ15N and δ18O. However, Δ17O indicated that about 20% of NO3- in TF derived from canopies nitrification in August, after a severe drought, with a lower proportion in September (≈ 8%). This seasonal partitioning between biologically and atmospherically derived NO3- coincided with a decreasing trend of the abundance of archaeal nitrifiers. Tree canopies and TF had more diverse microbial communities than RF. Yet, RF showed higher variability in microbial composition, likely associated to the origin of air masses. 4. Synthesis. Atmospheric N deposition is significantly altered after passing through tree canopies. While nitrification has been proposed as one of the mechanisms responsible for these changes, very few studies directly investigate its occurrence. Here, we showed that nitrification by epiphytic leaf microbes contributed to increasing NO3 in TF and that nitrifiers’ activity was reduced going from the dry and hot summer to the cool winter. Overall, these results highlight the power of coupling microbial community analysis, functional gene amplification and stable isotope approaches to examine ecosystem-scale processes.