Nitrate pollution in groundwater is a serious problem in many parts of the world. However, due to the diffuse and common spatially over-lapping character of potential several non-point pollution sources, it is often difficult to distinguish main nitrate sources responsible for the pollution. For this purpose, we present a novel methodology applied to groundwater for an intensely polluted area. Groundwater samples were collected monthly from April 2017 to March 2018 in Shimabara City, Nagasaki, Japan. Soil samples were collected seasonally at soil surface and 50 cm depth at 10 locations during the same period. Sequential extraction by water and extract agents was performed using calcium phosphate for anions and strontium chloride for cations. Mean nitrate concentration in groundwater close to a livestock waste disposal site (hereinafter called "LWDS") was 14.2 mg L-1, which is exceeding Japanese drinking water standards (10 mg L-1). We used coprostanol concentration, which is a fecal pollution indicator, to identify pollution sources related to livestock waste. For this purpose, we measured coprostanol (5β) and cholestanol (5α) and then calculated the sterol ratio (5β/(5β + 5α)). The ratios for three groundwater sampling sites were 0.28, 0.26, and 0.10, respectively. The sterol ratios indicated no pollution (< 0.3). However, the detection of coprostanol originating from animal and human waste showed that groundwater was clearly affected by this pollution source. Nitrate levels in the soil were relatively high in samples collected close to the LWDS and coprostanol contents were affected by livestock waste. Soil and groundwater nitrate concentrations displayed a complex but strong relationship. Nitrate contents were shown to be transported downstream from source areas in both soil and groundwater.