The Sürgü Stream, located in the Euphrates River basin of Turkey, is used for drinking water source, agricultural irrigation and rainbow trout production. Therefore, water quality of the stream is of great importance. In this study, multivariate statistical techniques (MSTs) and water quality index (WQI) were applied to assess water quality of the stream affected by multiple stressors such as untreated domestic sewage, effluents from fish farms, agricultural runoff and streambank erosion. For this, 16 water quality parameters at five sites along the stream were monitored monthly during one year. Most of parameters showed significant spatial variations, indicating the influence of anthropogenic activities. All parameters except TN (total nitrogen) showed significant seasonal differences due to high seasonality in WT (water temperature) and water flow. The spatial variations in the WQI were significant (p < 0.05) and the mean WQI values ranged from 87.6 to 95.3, indicating “good” to “excellent” water quality in the stream. Cluster analysis classified five sites into three groups, that is, clean region, low polluted region and very clean region. Stepwise temporal discriminant analysis (DA) identified that pH, WT, Cl−, SO42−, COD (chemical oxygen demand), TSS (total suspended solids) and Ca2+ are the parameters responsible for variations between seasons, and stepwise spatial DA identified that DO (dissolved oxygen), EC (electrical conductivity), NH4–N, TN (total nitrogen) and TSS are the parameters responsible for variations between the regions. Principal component analysis/factor analysis revealed that the parameters responsible for water quality variations were mainly associated with suspended solids (both natural and anthropogenic), soluble salts (natural) and nutrients and organic matter (anthropogenic).