Defaunation studies have shown that rumen protozoa are one of the main causes of low nitrogen utilization efficiency due to their bacterivory and subsequent intraruminal nitrogen recycling in ruminants. In genomic and transcriptomic studies, we found that rumen protozoa express lysozymes and peptidases at high levels. We hypothesized that specific inhibition of lysozyme and peptidases could reduce the activity and growth of rumen protozoa, which can decrease proteolysis and ammoniagenesis by rumen microbiota. To test the above hypothesis, we evaluated 3 specific inhibitors [imidazole (IMI), phenylmethylsulphonyl fluoride (PMSF) and iodoacetamide (IOD)], both individually and in combinations, of lysozyme and different types of peptidases, with sodium dodecyl sulfate as a positive control. Rumen fluid was collected from 2 Jersey cows fed either a concentrate-based dairy ration or alfalfa hay only. Each protozoa-enriched rumen fluid was incubated for 24 h with or without the aforementioned inhibitors and fed a mixture of ground wheat grain, alfalfa, and grass hays to support microbial growth. Live protozoa cells were morphologically identified and counted at 3, 6, 12 and 24 h of incubation. Fermentation characteristics and prokaryotic composition were determined at 24 h of incubation followed by statistical test using GLIMMIX of SAS. Except for IOD, all the inhibitors reduced all the 9 protozoal genera detected, but to different extents, in a time-dependent manner. IOD was the least inhibitory to protozoa, but it halved ammoniagenesis while not decreasing feed digestibility or VFA. ANCOM analysis identified loss of Fibrobacter and overgrowth of Succinivibrio and Treponema up to 43 and 21%, respectively. Functional prediction using the CowPI database showed that all the inhibitors decreased the relative abundance of the genes encoding lysosome and peptidases. Overall, inhibition of protozoa resulted in changes in prokaryotic microbiota and in vitro fermentation, and peptidases, especially cysteine-peptidase, may be targeted to improve nitrogen balance in the rumen.