Phasins, the major proteins coating polyhydroxyalkanoate (PHA) granules, have been proposed as suitable biosurfactants for multiple applications because of their amphiphilic nature. In this work, we analyzed the interfacial activity of the amphiphilic alpha-helical phasin PhaF from Pseudomonas putida KT2440 at different hydrophobic hydrophilic interfacial environments. The binding of PhaF to surfaces containing PHA or phospholipids, postulated as structural components of PHA granules, was confirmed in vitro using supported lipid bilayers and confocal microscopy, with polyhydroxyoctanoate-co-hexanoate P(HO-co-HHx) and Escherichia coli lipid extract as model systems. The surfactant like capabilities of PhaF were determined by measuring changes in surface pressure in Langmuir devices. PhaF spontaneously adsorbed at the air-water interface, reducing the surface tension from 72 mN/m (water surface tension at 25 degrees C) to 50 mN/m. The differences in the adsorption of the protein in the presence of different phospholipid films showed a marked preference for phosphatidylglycerol species, such as 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol. The PHA-binding domain of PhaF (BioF) conserved a similar surface activity to PhaF, suggesting that it is responsible for the surfactant properties of the whole protein. These new findings not only increase our knowledge about the role of phasins in the PHA machinery but also open new outlooks for the application of these proteins as biosurfactants.