Air composition influences Dry Eye (DE) symptoms as demonstrated by studies that have linked the outdoor environment to DE. However, there is insufficient data on the effect of short-term exposure to indoor environments on DE symptoms. We conducted a prospective experimental research, in which an older building served as an experimental site, and a newer building served as the control site. Indoor air quality was monitored in both buildings. One-hundred-and-ninety-four randomly selected individuals were interviewed in the afternoon exiting the buildings and de-identified responses were recorded. Self-reported DE symptoms were modeled with respect to experimental and control buildings, adjusting for potential confounders. The experimental site had 2-fold higher concentration of airborne particulate matter (24,436 vs. 12,213 ≥ 0.5 µm/ft3) and microbial colonies (1066 vs. 400/m3), as compared to the control building. DE symptoms were reported by 37.5% of individuals exiting the experimental and 28.4% exiting the control building. In the univariate analysis, subjects exiting the experimental building were 2.21× more likely to report worsening of DE symptoms since morning compared to the control building (p < 0.05). When adjusting for confounders, including a history of eye allergy, subjects from the experimental building were 13.3× more likely to report worsening of their DE symptoms (p < 0.05). Our findings suggest that short-term exposure to adverse indoor environmental conditions, specifically air pollution and bioaerosols, has an acutely negative impact on DE symptoms.