The plains on the lowlands surrounding Lake Biwa, Japan, have long been used for rice (Oriza sativa L.) cultivation. In this study, we investigate the fate of herbicides commonly used in this agroecosystem. During the spring, the highest concentrations of simetryn, mefenacet, bromobutide and dymron were detected on the lake’s surface and at the mid-depth (20 m), indicating that agricultural runoff enters the lake and directly affects its water quality. Our findings also suggest that these contaminants might serve as an indicator of the transport pathways of some agricultural pollutants into the lake.