To study the deterioration of the water quality in Lake Biwa, Japan, over the last 40 years, we measured the concentrations and isotopic ratios of sulfur and strontium of water in 41 inflowing rivers and one discharging river. The concentrations of SO4 and Sr of inflowing rivers at downstream sites were generally high in the southern urban area and in the eastern area, where a large agricultural plain is situated, but low in the northern and western areas, whose watersheds are mountainous and with low population density. SO4 and Sr concentrations are also lower at upstream sites, which are closer to mountainous areas. Thus, the inflowing river receives large amounts of SO4 and Sr as it flows across the plain, where human activity levels are high. The delta34S or 87Sr/86Sr values of most eastern rivers at downstream sites are lower than those of water in Lake Biwa, and values become more uniform as the proportion of the plain area in the watershed increases. River water in other areas has higher values of delta34S or 87Sr/86Sr than the lake water. This result indicates that the decadal decrease of delta34S and 87Sr/86Sr in the lake water has been caused mainly by the increased flux of SO4 and Sr from rivers in the eastern plain. We assume that in the plain, sulfur, nitrogen, and organic compounds induced by human activities generate sulfuric, nitric, and organic acids in the water, which accelerate the extraction of Sr from bedrocks, leading to the generation of Sr in the river water in the area.