The aims of the study were to investigate the concentration of trace elements, nutrients, and ecotoxicity in bottom sediment cores collected from the silted part of the Rożnów reservoir (Southern Poland). Significant differences in the content of nutrients, trace elements, and ecotoxicity between five sediment cores were found. However, in the vertical distribution, there was no high variability of the above parameters, which means that the intensely suspended matter transported by the Dunajec river is and, at various times, has been homogeneous. Significant correlations between nutrients and trace elements (r = 0.33–0.91, at p ≤ 0.05) point to the same sources of the above-mentioned substances and similar levels of contamination in the sediment cores. However, the PCA results showed that cadmium and phosphorus in the sediment cores had different behaviors than other elements and can be associated mainly with anthropogenic sources. According to the degree of contamination factor, sediment cores fall under the category of considerable contamination of metals. Geochemical factors indicated that nickel, chromium, and cadmium (only sediment core C1) were found to be the cause of significant pollution in the sediment cores. Toxicity assessment found that most of the bottom sediment samples were classified as non-toxic or slightly toxic, only 10% of the sediment samples were toxic for Vibrio fischeri, and 6% of the samples were toxic for Sinapis alba. The two test organisms showed a different sensitivity, and higher toxic responses were recorded for V. fischeri than for S. alba. Cadmium and phosphorus were associated with toxicity for S. alba (r = 0.29–0.58, at p ≤ 0.05), whereas TOC, N, and S, and Ca for stimulation of growth this plants. Trace elements (r = 0.32–0.51, at p ≤ 0.05) and nutrients (S, K, Mg, Na, r = 0.44–0.58, at p ≤ 0.05) were positively correlated with inhibition of luminescence of V. fischeri. The studies of concentration and relation between trace elements, nutrients, and ecotoxicity are important in the ecological risk assessment and describing the quality of sediments with multiple sources contamination.