Nitrapyrin is a nitrification inhibitor used to retain ammonia-N in soil to improve crop yields and quality. Nitrapyrin targets specifically the ammonia oxidizers, but it is not known if it has non-target effects on the soil microbial communities. Here, we tested the hypothesis that nitrapyrin also leads to large shifts in soil microbial community structure, composition, diversity and functions. To test this hypothesis, we set-up a field experiment where wheat (Triticum aestivum cv. AC Walton) was fertilized with ammonium nitrate (NH4NO3) and supplemented or not with nitrapyrin. Rhizosphere and bulk soils were sampled twice (at grain filling and harvest), the 16S rRNA gene and ITS region were amplified and sequenced to follow the changes in archaeal, bacterial and fungal community structure, composition and diversity. To assess microbial functions, several genes involved in the nitrogen cycle were quantified by real-time qPCR, and volatile organic compounds (VOCs) were trapped in the rhizosphere at the moment of sampling. Sampling dates and soil compartments had overwhelming effects on the microbial communities. However, nitrapyrin still significantly affected the relative abundance of Thaumarchaeota, Proteobacteria, Nitrospirae and Basidiomycota, and several genera. Nitrapyrin also significantly affected bacterial and fungal community structure, and the abundance of all the N-cycle gene tested, but always in interaction with the sampling date. In contrast, nitrapyrin had no significant effect on the emission of VOCs, where only sampling dates significantly influenced the profiles observed. Our results show that nitrapyrin influences non-target soil- and plant-associated microbial communities. In the longer term, these shifts might counteract the positive effect of nitrapyrin on crop nutrition and greenhouse gas emissions.