In this manuscript, samples of Kupa River sediments were examined using three different extraction agents. The aim of this study was to evaluate the applicability of single extraction procedures to investigate the bioavailability and mobility of major and trace elements (Al, As, Ba, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, P, Pb, S, Si, Sr, Ti, V, and Zn) from river sediment. Two forms of studied elements were evaluated: mobile, the most toxic element form (extraction with 1 M CH3COONH4 and 0.01 M CaCl2) and potentially mobilized form (2 M HNO3 extraction). The estimation of the ecological risk, with the application of the probability distribution of RI (potential ecological risk index) values, is yielded with the help of the Monte Carlo simulation (MCS). Ammonium acetate is proved to be a better extraction agent than calcium chloride. A positive correlation between the content of all extracted elements with nitric acid and the total element content indicates that 2 M HNO3 efficiently extracts all studied elements. Results showed anthropogenic sources of cadmium and copper and high barium mobility. The MCS suggests that risk of Cr, Cu, Ni, Pb, and Zn was low; As and Cd posed a lower and median ecological risk in the studied areas.