The use of surfactants as drug permeability enhancers across epithelial barriers remains a challenge. Although many studies have been performed in this field using synthetic surfactants, the possibility of employing surfactants produced by bacteria (the so called biosurfactants”) has not been completely explored. Among them, one of the most well characterized class of biosurfactants are rhamnolipids. The aim of the study was to investigate the effect of rhamnolipids on the epithelial permeability of fluorescein isothiocyanate-labelled dextrans 4 kDa and 10 kDa (named FD4 and FD10, respectively) as model for macromolecular drugs, across Caco-2 and Calu-3 monolayers. These cell lines were selected as an in vitro model for the oral and respiratory administration of drugs. Before performing permeability studies, rhamnolipids mixture was analysed in terms of chemical composition and quantification through mass analysis and HPLC. Cytotoxicity and transepithelial electrical resistance (TEER) studies were also conducted using Caco-2 and Calu-3 cell lines. A dose-dependent effect of rhamnolipids on TEER and FD4 or FD10 permeability across both cell lines was observed at relatively safe concentrations. Overall, results suggest the possibility of using rhamnolipids as absorption enhancers for macromolecular drugs through a reversible tight junction opening (paracellular route), despite more investigations are required to confirm their mechanism of action in term of permeability.