Despite the wide interest raised by lung administration of nanoparticles (NPs) for the treatment of various diseases, little information is available on their effect toward the airway epithelial barrier function. In this study, the potential damage of the pulmonary epithelium upon exposure to poly(lactide-co-glycolide) (PLGA) NPs has been assessed in vitro using a Calu-3-based model of the bronchial epithelial barrier. Positively and negatively charged as well as neutral PLGA NPs were obtained by coating their surface with chitosan (CS), poloxamer (PF68), or poly(vinyl alcohol) (PVA). The role of NP surface chemistry and charge on the epithelial resistance and mucus turnover, using MUC5AC as a marker, was investigated. The interaction with mucin reduced the penetration of CS- and PVA-coated NPs, while the hydrophilic PF68-coated NPs diffused across the mucus barrier leading to a higher intracellular accumulation. Only CS-coated NPs caused a transient but reversible decrease of the trans-epithelial electrical resistance (TEER). None of the NP formulations increased MUC5AC mRNA expression or the protein levels. These in vitro results highlight the safety of PLGA NPs toward the integrity and function of the bronchial airway barrier and demonstrate the crucial role of NP surface properties to achieve a controlled and sustained delivery of drugs via the pulmonary route.