Freshwater biodiversity is declining faster than marine or terrestrial diversity, yet its drivers are much less known. Although dams were shown to negatively affect river habitats, fragmentation by bridges has received less attention and is not as well understood. We tested whether and how bridges present barriers to aquatic insects by studying mass swarmings of Palingenia longicauda mayflies on river Tisza (NE-Hungary). Behavioural observations showed that upon approaching the bridge, upstream-flying mayflies typically turned back and 86% of them never crossed the bridge. Lack of physical contact showed that the bridge was an optical, rather than a mechanical barrier for the polarotactic mayflies. Imaging polarimetry showed that the bridge disrupted the horizontally polarizing channel guiding the flight of mayflies above the river. Energy loss, demonstrated by calorimetry, and time constraints forced females to lay eggs only downstream from the bridge. Counts of larval skins shed by swarming individuals showed nearly 2 to 1 female per male downstream from the bridge, while sex ratio above the bridge was slightly male-biased. We suggest that the surplus of parthenogenetic females, that produce only female larvae, downstream from the bridge may have led to the observed sex-ratio bias since the construction of the bridge (1942). Our results demonstrate that bridges can be optical barriers for aquatic insects and can cause population-level impacts, such as biased sex ratios, in natural populations. Sex ratio biases due to bridges may decrease effective population size and genetic variability, which may have contributed to the recent extinction of this species from most of Europe.