The impact of DMPP (3,4-dimethylpyrazole phosphate), applied at two doses (low: recommended for agronomic use; high: > 100 × the recommended), on the function, diversity, and dynamics of target microorganisms (ammonia-oxidizing microorganisms, AOM), functionally associated microorganisms (nitrite-oxidizing bacteria (NOB) and denitrifiers), and total prokaryotic and fungal microbial communities was assessed in two loamy soils, mainly differing in pH (acidic vs. alkaline), in a 35-day microcosm study. This was achieved via monitoring inorganic N-pools, potential nitrification (PN) rates, amoA gene and transcripts abundance, the abundance of other phylogenetic marker genes (nxrB, narG, nirS, nirK, nosZ, 16S rRNA, 18S rRNA), and amplicon sequencing of amoA, 16S rRNA, and ITS. Overall, DMPP was more persistent in the acidic soil. Its low dose successfully inhibited nitrification in the alkaline but not in the acidic soil, where effective inhibition was observed only at the high dose. This was mainly attributed to the consistently higher activity of DMPP towards ammonia-oxidizing bacteria (AOB) prevailing in the alkaline soil, unlike ammonia-oxidizing archaea (AOA) whose abundance and transcriptional activity was reduced only by the high dose. DMPP, at the high dose, reduced the abundance of Nitrobacter but not Nitrospira NOB, while its low dose increased the abundance of denitrifying bacteria, prokaryotic, and fungal populations in the alkaline soil. Amplicon sequencing revealed that DMPP imposed significant changes in the composition of the prokaryotic, fungal, and AOB communities in both soils, unlike AOA which were less responsive. These were associated with dose-dependent changes in the abundance of bacteria and fungi known to control key soil functions implying possible effects for the soil ecosystem homeostasis. Our study paves the way for a more comprehensive analysis of the effects of NIs on the soil microbial community, beyond the current focus on target AOM.