The tropical pasture grass, Brachiaria humidicola (Rendle) Schweick, produces nitrification inhibitory compounds (termed biological nitrification inhibitors or BNIs) in its shoot and root tissues and releases BNIs from its roots. In the present study, two BNI compounds were isolated and identified from the shoot tissue of B. humidicola using activity-guided fractionation. The recombinant Nitrosomonas europaea containing luxAB genes derived from the bioluminescent marine gram-negative bacterium Vibrio harveyi, were used to determine BNI activity. The BNI compounds in the shoot tissue were identified as linoleic acid (LA) and linolenic acid (LN) using authentic-chemicals obtained from ©Sigma (ED80 16.0 μg ml−1 for both LA and LN) for verification. None of the other tested free fatty acids namely stearic acid, oleic acid, arachidonic acid, and cis-vaccenic acid showed any inhibitory effect on nitrification. Among the fatty acid methyl esters (FAME) evaluated [methyl oleate, methyl linoleate (LA-ME) and methyl linoleneate (LN-ME)], only LA-ME showed an inhibitory effect (ED80 8.0 μg ml−1). The inhibitory effect of LA, LN and LA-ME in the soil was stable for 120 days at 20°C. Soil treated with LA, LN and LA-ME showed a very low accumulation of NO3− and the maintenance of soil inorganic N in the NH4+ form. The inhibitory effect of LA-ME on soil nitrification was greater than that of LA or LN. In addition to BNI activity, both LA and LA-ME showed a suppressive effect on urea hydrolysis in soil. Both LA and LN blocked the AMO (ammonia monooxygenase) and HAO (hydroxylamino oxidoreductase) enzymatic pathways in Nitrosomonas. Since LA and LN can be produced from vegetable oils such as soybean, flax or sunflower, they have the potential for use as nitrification inhibitors in production agriculture.