Nitrification is associated with significant losses of fertilizer-derived ammonium from agroecosystems. The use of biological nitrification inhibitors (BNIs), in place of synthetic nitrification inhibitors (SNIs), holds a great potential to effectively reduce N losses and conforms with the current move towards ecological-intensified agriculture. Knowledge of the activity of BNIs to soil nitrifiers is limited and is mostly based on bioassays with a single Nitrosomonas europaea strain. We determined the in vitro activity of multiple plant-derived compounds as BNIs like (i) root-derived compounds (sakuranetin, MHPP, and zeanone); (ii) other phytochemicals (caffeic acid, quinic acid, chlorogenic acid and shikimic acid), and (iii) analogues of statins (simvastatin), triazoles (1-butyl-4-propyl-triazole, 1,4-dibutyltriazole) and zeanone (2-methoxy-1,4-naphthoquinone), on ecophysiologically and phylogenetically distinct soil-derived ammonia-oxidizing bacteria (AOB) (Nitrosospira multiformis and N. europaea), ammonia-oxidizing archaea (AOA) (Candidatus Nitrosotalea sinensis and Candidatus Nitrosocosmicus franklandianus), and a nitrite-oxidizing bacterium (NOB) (Nitrobacter sp. NHB1). AOA were more sensitive than AOB to BNIs. Sensitivity within AOA group was BNI-dependent, unlike AOB for which N. multiformis was consistently more sensitive than N. europaea. Interestingly, several compounds were inhibitory to Nitrobacter sp. with MHPP and caffeic acid being more potent to the NOB compared to the AOB and AOA strains, an observation with potentially serious implications for soil quality and agricultural production. Overall, zeanone, MHPP and caffeic acid were the most potent ΒNIs towards AOB, zeanone and 2-methoxy-1,4-naphthoquinone were the most effective compounds against AOA, while caffeic acid was the most potent BNI on Nitrobacter sp. We provide pioneering evidence for the activity range of multiple BNIs on soil nitrifiers, stress the need for revisiting the biological screening systems currently used for BNI determination and we sought for a more thorough monitoring of the impact of BNI candidates on a range of both target and non-target microorganisms.