Chemical compounds, such as the CS gas employed in military operations, have a number of characteristics that impact the ecosystem by upsetting its natural balance. In this work, the toxicity limit and microorganism’s reaction to the oxidative stress induced by O-chlorobenzylidenemalonitrile, a chemical found in CS gas, were assessed in relation to the green algae Chlorella pyrenoidosa. A number of parameters, including the cell growth curve, the percent inhibition in yield, the dry cell weight, the percentage viability and productivity of algal biomass flocculation activity, and the change in oxygen production, were analyzed in order to comprehend the toxicological mechanisms of O-chlorobenzylidenemalonitrile on algal culture. Using fluorescence and Fourier transform infrared spectroscopy (FTIR), the content of chlorophyll pigments was determined. The values obtained for pH during the adaptation period of the C. pyrenoidosa culture were between 6.0 and 6.8, O2 had values between 6.5 and 7.0 mg/L, and the conductivity was 165–210 µS/cm. For the 20 µg/mL O-chlorobenzylidenemalonitrile concentration, the cell viability percentage was over 97.4%, and for the 150 µg/mL O-chlorobenzylidenemalonitrile concentration was 74%. The ECb50 value for C. pyrenoidosa was determined from the slope of the calibration curve; it was estimated by extrapolation to the value of 298.24 µg/mL. With the help of this study, basic information on the toxicity of O-chlorobenzylidenemalonitrile to aquatic creatures will be available, which will serve as a foundation for evaluating the possible effects on aquatic ecosystems. The management of the decontamination of the impacted areas could take the results into consideration.