The application of combined research methods, such as hydrochemical and isotopic analyses, facilitates understanding of the origin of water constituents and migration of contaminants in the aquatic environment. The presented study attempts to identify contamination sources affecting water quality within the Kozłowa Góra reservoir catchment area (southern Poland). A total of 100 water samples were collected from surface water (the Brynica river, streams, and the reservoir) and groundwater (a Quarternary aquifer) in four time periods. During fieldwork, the physicochemical parameters were measured (temperature, pH, electrical conductivity, redox potential, and dissolved oxygen). The chemical analysis included the determination of major ions (HCO3−, SO42−, Cl−, Ca2+, Mg2+, Na+, and K+), nutrients (NO3−, NO2−, NH4+, and PO43−), and total organic carbon. The study was complemented by isotopic analysis of sulphur and oxygen in sulphate, which enabled a more precise identification of stressors affecting water quality in different parts of the catchment area. Chemical and isotopic results (δ34S = 4.38–13.99‰; δ18O 3.59–13.30‰) revealed that wastewater discharges and agricultural activities have a significant influence on the chemistry of the Brynica River and other streams. At some sampling points, a lower quality of water was manifested by elevated concentrations of NO3− (up to 22.6 mg/L) and several other ions (e.g., up to 114 mg/L for Cl−, up to 51.52 mg/L for NH4+, and 12.5 mg/L for PO43−). The quality of groundwater was deteriorated mainly by infiltration of sewage from leaky septic tanks. The level of groundwater contamination varied depending on the location, as higher concentrations of major ions and values of electrical conductivity were observed in residential areas. The Brynica river, streams, and groundwater recharge the drinking water reservoir, and thus its quality depends on these sources. A dilution of water and the presence of biochemical processes led to a reduction of contaminant concentrations in the Kozłowa Góra reservoir (down to 3.5 mg/L for NO3−, 32 mg/L for Cl−, 0.21 mg/L for NH4+, and <0.05 mg/L for PO43−) compared to water in the river and streams supplying the reservoir. The study revealed the role of wastewater discharge and agricultural activities in the evolution of surface water chemistry. The results will be used in further research on the origin and migration of other substances in water, including microcontaminants.