Differentiated primary human respiratory epithelial cells grown at air-liquid interface have become a widely used cell culture model of the human conducting airways. These cultures contain secretory cells such as goblet and club cells, which produce and secrete mucus. Here, we characterize the composition of mucus harvested from airway cultures of nasal and bronchial origin. We find that despite inter-donor variability, the salt, sugar, lipid, and protein content and composition are very similar between nasal and bronchial mucus. However, subtle differences in the abundance of individual components in nasal versus bronchial mucus can influence its antimicrobial properties. The ability of mucus to neutralize influenza A virus varies with the anatomical origin of the airway cultures and correlates with the abundance of triglycerides and specific sialylated glycoproteins and glycolipids.