Purpose <p>This study aimed to verify the association of CO₂ concentration and mood in people performing exercise in gyms. Methods: A total of 327 exercise practitioners (149 men and 178 women) were evaluated, with a mean age of 35.1&#xa0;years, exercising at 3 gyms ventilated with either split-system or central system air conditioners. The air quality was assessed by determining the CO₂ concentration level, as measured by a non-dispersive infrared sensor, positioned centrally in the weight room, at a height that corresponds to the breathing zone, measuring continuously from 7:00 a.m. to 11:00 p.m. CO₂ measurements were conducted for 1&#xa0;week in each gym. Mood states were evaluated using the Brunel Mood Scale. Results: The recommended value for indoor CO₂ level according to the national guideline of Brazil (1000&#xa0;ppm) was exceeded for 79.6% of the time. The highest mean CO₂ concentration (<i>p</i> &lt; 0.01) and the maximum value were measured in gym 1, which was ventilated with a split system. Exercise practitioners in gyms with CO₂ concentrations below this value showed higher vigor than those in gyms above 1000&#xa0;ppm (<i>p</i> = 0.02). Binary logistic regression revealed that exercise practitioners in gyms with high CO₂ concentrations (above 2146&#xa0;ppm) were 2.26 times more likely to present a negative mood disturbance (<i>p</i> = 0.01) than those in gyms with up to 1115&#xa0;ppm. Conclusion: We concluded that there was an association of CO₂ concentration and mood in people performing exercise in gyms. Increasing the natural ventilation through opening doors and windows is suggested as practical implication.</p>