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Inhibition of ruminal hyper-ammonia-producing bacteria (HAB) is gaining importance in recent days for reducing degradation of dietary proteins in the rumen aimed at effective protein utilization and reducing environmental pollution through the excretion of fecal nitrogen. This study examined the efficacy of bioactive plant extracts and essential oil compounds in the inhibition of ruminal HAB enrichment culture from buffalo with the aim of developing a feed additive for effective protein utilization and reducing environmental pollution. HAB culture from buffalo rumen was prepared in an enrichment medium. In vitro incubation of graded dose levels of various essential oils and plant extracts in HAB enrichment culture was steered and inhibition of deaminase activity of HAB was examined by reduced ammonia production, protease activity and bacterial growth. The dosing of garlic oil, origanum oil and thymol demonstrated a reduction (p < 0.05) in ammonia production at 12 h and 24 h of incubation, irrespective of doses. Although origanum oil was effective in reducing (p < 0.05) protease activity, quillaza bark saponin, garlic oil, thymol and extracts of Psidium guajava, Cannabis sativa, and Allium schoenoprasum were futile (p > 0.05) in reducing protease concentration. The HAB bacterial growth was also inhibited (p < 0.05) by origanum oil, irrespective of dose levels, however, eugenol was effective (p < 0.05) in the highest (0.75 g/l) examined dose only. Therefore, the study revealed a reduction of ammonia and protease production through HAB inhibition by bioactive plant compounds based on metabolites and their dose. Origanum oil administration resulted significant inhibition of HAB growth, ammonia production, and protease activity in enrichment culture indicating its high potential for application as a feed additive to enhancing protein utilization by ruminant animals and reducing environmental pollution. However, corroboration warrants in vivo trial in growing animals for nitrogen balance study before practical application in ruminants' diet.