The global use of nitrogen (N) fertilizer has increased 10-fold in the last fifty years, resulting in increased N losses via nitrate leaching to groundwater bodies or from gaseous emissions to the atmosphere. One of the biggest problems farmers face in agricultural production systems is the loss of N. In this context, novel biological nitrification inhibitors (BNI) using biochar (BC) as a renewable matrix to increase N use efficiency, by reducing nitrification rates, have been evaluated. The chemical and morphological characteristics of BC were analyzed and BC-BNI complexes were formulated using plant extracts from pine (Pinus radiata), eucalyptus (Eucalyptus globulus) and peumo (Cryptocarya alba). In field experiments, fertilizer and treatments, based on crude plant extracts and BC-BNI complexes, were applied and the effect on nitrification was periodically monitored, and at the laboratory level, a phytotoxicity assay was performed. The biochar-peumo (BCPe) complex showed the highest nitrification inhibition (66%) on day 60 after application compared with the crude plant extract, suggesting that BCPe complex protects the BNI against biotic or abiotic factors, and therefore BC-BNI complexes could increase the persistence of biological nitrification inhibitors. None of the biochar complexes had toxic effect on radish plants.