Heavy metal pollution is still present in the Danube River basin, due to intensive naval and agricultural activities conducted in the area. Therefore, continuous monitoring of this pivotal aquatic macro-system is necessary, through the development and optimization of monitoring methodologies. The main objective of the present study was to develop a prediction model for heavy metals accumulation in biological tissues, based on field gathered data which uses bioindicators (fish) and oxidative stress (OS) biomarkers. Samples of water and fish were collected from the lower sector of Danube River (DR), Danube Delta (DD) and Black Sea (BS). The following indicators were analyzed in samples: cadmium (Cd), lead (Pb), iron (Fe), zinc (Zn), copper (Cu) (in water and fish tissues), respectively, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) (in fish tissues). The pollution index (PI) was calculated to identify the most polluted studied ecosystem, which revealed that Danube River is seriously affected by the presence of Fe (IP = 4887) and strongly affected by the presence of Zn (IP = 4.49). The concentration of Cd in fish muscle tissue was above the maximum permitted level (0.05 µg/g) by the EU regulation. From all analyzed OS biomarkers, MDA registered the highest median values in fish muscle (145.7 nmol/mg protein in DR, 201.03 nmol/mg protein in DD, 148.58 nmol/mg protein in BS) and fish liver (200.28 nmol/mg protein in DR, 163.67 nmol/mg protein, 158.51 nmol/mg protein), compared to CAT, SOD and GPx. The prediction of Cd, Pb, Zn, Fe and Cu in fish hepatic and muscle tissue was determined based on CAT, SOD, GPx and MDA, by using non-linear tree-based RF prediction models. The analysis emphasizes that MDA in hepatic tissue is the most important independent variable for predicting heavy metals in fish muscle and tissues at BS coast, followed by GPx in both hepatic and muscle tissues. The RF analytical framework revealed that CAT in muscle tissue, respectively, MDA and GPx in hepatic tissues are most common predictors for determining the heavy metals concentration in both muscle and hepatic tissues in DD area. For DR, the MDA in muscle, followed by MDA in hepatic tissue are the main predictors in RF analysis.