Urine from sheep fed with different plantain cultivars (Plantago lanceolata L.) was characterised to determine if metabolites in the urine contributed to soil biological nitrification inhibition (BNI) activity. Chromatographic analyses, combined with mass spectrometry, revealed there were more than 800 mass features that were unique to, or present in much greater concentration, in urine from sheep fed plantain than in urine of ryegrass (Lolium perenne L.)-fed sheep. The plantain metabolites aucubin, verbascoside, and catalpol have been implicated in relation to BNI activity, but these compounds were not detected in the urine of sheep fed with plantain. However, based on the mass spectral fragmentation patterns, metabolic derivatives of these compounds were present. A series of incubation studies showed that short-term nitrification inhibition did occur when plantain urine was applied to soil. Inhibition depended on several factors, including length of plantain grazing period and plantain cultivar type. As the incubations progressed, the inhibitory effect declined, potentially because of degradation of the inhibitory compounds by soil microorganisms. Urine of sheep fed with the plantain cultivar ‘Agritonic’ had a particularly strong inhibitory effect, which persisted for more than 1 month at an incubation temperature of 20 °C. Furthermore, in a laboratory bioassay using a pure culture of ammonia-oxidising bacteria, the organic fraction of urine from sheep fed with ‘Agritonic’ plantain was shown to be a strong nitrification inhibitor. This work suggests that valuable environmental benefits can be obtained by including cultivars of plantain showing BNI activity in pastures.