A portable voltammetric sensor for toxicity assessment was developed based on a screen-printed carbon electrode modified with SiO₂-CuO-glucose oxidase (GOx) film. The method is based on the detection of the metabolic activity of E. coli towards glucose as an indicator systems in the presence of antiseptic drugs as model toxic compounds. CuO particles exhibited catalytic activity toward hydrogen peroxide produced by the GOx-induced oxidation of glucose. A well-defined reduction peak was registered at E=−0.50 V (vs. Ag/AgCl). The metabolism of glucose by E. coli resulted in a decrease in analytical signal of glucose with the increasing bacteria content in the range of 0.8×10⁹–5.0×10⁹ CFU mL⁻¹ with a calculated limit of detection (LOD) of 0.59×10⁹ CFU mL⁻¹. To evaluate the effect of antiseptics the biosensor was tested in E. coli solution with chlorhexidine (CHD) or decamethoxine (DMT). A linear dependence of glucose consumption by E. coli solution on the antiseptic concentration was obtained in the range of 3.0–12.0 μg mL⁻¹ for CHG and 1.0–12.0 μg mL⁻¹ for DMT, respectively. The developed third generation biosensor showed satisfactory stability and reproducibility of the analytical response. A modified electrode can be used for at least two months.