To control agronomic N losses and reduce environmental pollution, biological nitrification inhibition (BNI) is a promising strategy. BNI is an ecological phenomenon by which certain plants release bioactive compounds that can suppress nitrifying soil microbes. Herein, we report on two hydrophobic BNI compounds released from maize root exudation (1 and 2), together with two BNI compounds inside maize roots (3 and 4). On the basis of a bioassay-guided fractionation method using a recombinant nitrifying bacterium Nitrosomonas europaea, 2,7-dimethoxy-1,4-naphthoquinone (1, ED50 = 2 μM) was identified for the first time from dichloromethane (DCM) wash concentrate of maize root surface and named “zeanone.” The benzoxazinoid 2-hydroxy-4,7-dimethoxy-2H-1,4-benzoxazin-3(4H)-one (HDMBOA, 2, ED50 = 13 μM) was isolated from DCM extract of maize roots, and two analogs of compound 2, 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (HMBOA, 3, ED50 = 91 μM) and HDMBOA-β-glucoside (4, ED50 = 94 μM), were isolated from methanol extract of maize roots. Their chemical structures (1–4) were determined by extensive spectroscopic methods. The contributions of these four isolated BNI compounds (1–4) to the hydrophobic BNI activity in maize roots were 19%, 20%, 2%, and 4%, respectively. A possible biosynthetic pathway for zeanone (1) is proposed. These results provide insights into the strength of hydrophobic BNI activity released from maize root systems, the chemical identities of the isolated BNIs, and their relative contribution to the BNI activity from maize root systems.