Targeted strategies to overcome defects in organic semiconductors require insight into their identity and origin. Here the formation, nature, and location of defects is studied in PM6:Y6 organic solar cells by sensitive EQE measurements. Exposure of the active layer to ambient atmosphere and H2O‐saturated compressed air indicates that a trace constituent in ambient air causes the formation of defects. By exposing the active layer to O3‐enriched air, O3 is identified as the species creating defects in PM6:Y6 blends. Aging of complete inverted (n–i–p) configuration solar cells in H2O‐saturated compressed air also increases the defect response. This is attributed to a reduced band bending at the PM6:Y6 | MoO3 hole‐collecting contact, caused by a change in work function of MoO3 interacting with the H2O, which allows more defect states to be filled and available for photoexcitation. By measuring energy resolved‐electrochemical impedance spectroscopy and by fabricating semitransparent cells, regular architecture cells, and semitransparent cells with an optical spacer−mirror stack it is found that defects originate predominantly from PM6 and are located near the top electrode, independent of device polarity. Because O3 is omnipresent in ambient atmosphere, albeit in small amounts, it likely causes defects in many organic semiconductors exposed to ambient air. Sensitive external quantum efficiency measurements combined with energy resolved‐electrochemical impedance spectroscopy reveal that ozone, and not water or oxygen, in ambient air is the origin of sub‐bandgap defects that occur in polymer donor–non‐fullerene acceptor organic solar cells. The defects are primarily located at the top side of the film and arise due to degradation of the polymer donor.