The aim of current paper was to investigate the possibility of increasing the toxicity of calcium hypochlorite (Ca(ClO)2) and hydrogen peroxide (H2O2) onEscherichia coli K-12by preliminary enrichment of culture media by carbon dioxide (CO2). For this purpose, the microbes sensitivity to H2O2or/and Ca(ClO)2at normal and CO2-enriched medium was studied by spectrophotometric, radioisotopic, and electronmicroscopic methods. Ten-minute preincubation in CO2-enriched medium enhanced the toxic effect of both H2O2or/and Ca(ClO)2on bacteria as a result of induced growth inhibition, compared to no-CO2enriched group. Additionally, changes in cell morphology and proliferation were observed. It was demonstrated that the preliminary incubation of microbes in CO2-enriched culture media in nonsupercritical concentration elevate the toxic effect of H2O2or/and Ca(ClO)2on microbes. This can serve as a novel, effective, inexpensive, and environmentally friendly approach for water purification from bacteria, further improving the protection of the environment and human health.