Drosera rotundifolia has been traditionally used for the treatment of respiratory diseases in phytotherapy and homeopathy. The mechanisms of action recognized so far are linked to the known effects of specific components, such as flavonoids, but are not completely understood. In this study, the biological functions of D. rotundifolia were explored in vitro following the treatment of bronchial epithelial cells, which are the potential targets of the pharmacological effects of the herbal medicine. To do so, the whole plant ethanolic extract was 1000-fold diluted in water (D. rotundifolia 3×) and added to a 16HBE human cell line culture for 3 h or 6 h. The effects on gene expression of the treatments and corresponding controls were then investigated by RNA sequencing. The differentially expressed genes were validated through RT-qPCR, and the enriched biological functions involved in the effects of treatment were investigated. D. rotundifolia 3× did not impair cell viability and was shown to be a stimulant of cell functions by regulating the expression of dozens of genes after 3 h, and the effects were amplified after 6 h of treatment. The main differentially expressed genes encoded ligands of epithelial growth factor receptor, proteins involved in xenobiotic detoxification and cytokines, suggesting that D. rotundifolia 3× could stimulate self-repair systems, which are impaired in airway diseases. Furthermore, D. rotundifolia 3× acts on a complex and multifaceted set of genes and may potentially affect different layers of the bronchial mucosa.