Objectives The marine benthic nitrogen cycle is affected by both the presence and activity of macrofauna and the diversity of N-cycling microbes. However, integrated research simultaneously investigating macrofauna, microbes and N-cycling is lacking. We investigated spatio-temporal patterns in microbial community composition and diversity, macrofaunal abundance and their sediment reworking activity, and N-cycling in seven subtidal stations in the Southern North Sea. Spatio-Temporal Patterns of the Microbial Communities Our results indicated that bacteria (total and beta-AOB) showed more spatio-temporal variation than archaea (total and AOA) as sedimentation of organic matter and the subsequent changes in the environment had a stronger impact on their community composition and diversity indices in our study area. However, spatio-temporal patterns of total bacterial and beta-AOB communities were different and related to the availability of ammonium for the autotrophic beta-AOB. Highest bacterial richness and diversity were observed in June at the timing of the phytoplankton bloom deposition, while richness of beta-AOB as well as AOA peaked in September. Total archaeal community showed no temporal variation in diversity indices. Macrofauna, Microbes and the Benthic N-Cycle Distance based linear models revealed that, independent from the effect of grain size and the quality and quantity of sediment organic matter, nitrification and N-mineralization were affected by respectively the diversity of metabolically active beta-AOB and AOA, and the total bacteria, near the sediment-water interface. Separate models demonstrated a significant and independent effect of macrofaunal activities on community composition and richness of total bacteria, and diversity indices of metabolically active AOA. Diversity of beta-AOB was significantly affected by macrofaunal abundance. Our results support the link between microbial biodiversity and ecosystem functioning in marine sediments, and provided broad correlative support for the hypothesis that this relationship is modulated by macrofaunal activity. We hypothesized that the latter effect can be explained by their bioturbating and bio-irrigating activities, increasing the spatial complexity of the biogeochemical environment.