Abstract The aim of this work is to study the antifouling performance and water uptake behaviour of coatings formulated with papain (an environmentally friendly pigment). Antifouling coatings have been formulated using rosin (natural resin) as matrix and papain adsorbed in activated carbon as pigment. Electrochemical impedance spectroscopy (EIS) measurements were used to evaluate the behaviour of the formulated coatings in the marine environment and to calculate the apparent water coefficient of diffusion (D). FTIR and XPS analyses confirm the presence of papain adsorbed inside the activated carbon pores and the release of papain in water. Immersion tests in the Mediterranean Sea were carried out for 7 months to verify the degree of biofouling of the tested coatings. These field assays clearly indicate the excellent behaviour of papain-based antifouling coatings; the results being similar to those achieved using a commercial coating. Additionally, the EIS technique is shown to be a great tool to predict the coating diffusivity of antifouling coatings before immersion tests. Furthermore, the use of biodegradable papain as a nature-friendly antifouling agent can eliminate the negative environmental impact caused by metals and chemical biocides typically used in current commercial formulations.