Cognitive performance is essential to foster learning. High CO2 concentrations are common in classrooms and can lead to reduced cognitive performance. Negative air ions (NAIs) can improve cognitive performance. This study explored the effects of indoor NAIs on the cognitive performance and health of college students exposed to a high pure CO2 environment. Forty college students were exposed to four sets of conditions (NAIs+500 ppm CO2, 500 ppm CO2, NAIs+2500 ppm CO2, and 2500 ppm CO2). Participants’ cognitive performance, including reasoning, short-term memory, concentration, and verbal ability, was assessed under each condition using the Cambridge Brain Sciences tool. Acute health symptoms were investigated using a subjective questionnaire, and simultaneously, participants’ blood pressure, heart rate, and lung function were tested. Analysis of variance (ANOVA) in a repeated-measures design was used to analyze the effects of different conditions on cognitive performance and health symptoms. The results revealed that the different levels of CO2 and NAIs had a significant effect on cognitive performance after one hour of exposure and had no significant effect after three hours of exposure. Compared with 500 ppm CO2, 500 ppm CO2+NAIs resulted in better reasoning skills, short-term memory, and verbal skills, and 2500 ppm CO2 led to poorer reasoning skills. The addition of NAIs to 2500 ppm CO2 improved reasoning skills, short-term memory, and verbal skills. The benefits of adding NAIs to high pure CO2 condition on cognitive performance are more noticeable than those to low CO2 condition. Moreover, adding NAIs can reduce nasal irritation or dryness, skin irritation or dryness, sleepiness symptoms, and heart rate elevation caused by pure CO2. However, the benefits of NAIs on health symptoms and physiology were not observed under the 500 ppm CO2 condition. Adding NAIs to a high pure CO2 level is an effective means to improve the cognitive performance and health of college students.