The behavioral (filtration activity) and biochemical (oxidative stress) effects of Roundup 360 Plus (active substance glyphosate) herbicide on two species of unionid mussels, Unio tumidus (Philipsson, 1788) and Anodonta anatina (L.), were evaluated at concentrations ranging from 15 to 1500 μg L−1 of glyphosate for five days. During all experiments, we did not record the mortality of the studied mussel species. Exposure to Roundup herbicide induced dose-dependent filtration disruptions in both U. tumidus and A. anatina. Exposure of the mussels to a low and environmentally relevant concentration 15 µg glyphosate L−1 resulted in a slight (<20%) and temporary decrease in mean valve dilation. Exposure of the mussels to Roundup at relatively high concentrations caused drastic and prolonged shell closure and a reduction in the mussel shell opening rate. Exposure of both mussel species to herbicide resulted in oxidative stress; an increase in superoxide dismutase enzymatic activity was detected. The most significant increase in SOD activity was observed after the exposure to the highest Roundup concentration. However, no correlation between the Roundup concentration and enzymatic activity was found. The use of unionid mussels to detect environmentally relevant concentrations of Roundup, as a part of biological early warning system for pollution, is limited, but they can serve to detect the incidental pollution of aquatic ecosystems with high concentrations of this herbicide.