Abstract Background Biocides, in touch with the mucosa or skin, are capable of penetrating the mucosa or skin and causing possible local lesions, such as irritations, allergic contact dermatitis and even systemic effects such as carcinogenesis. The high permeability of compounds across the oral or nasal mucosa is widely known, in comparison to the poor penetration across the skin. Therefore, the main objective of this work is to produce a formulation that can strengthen the mucosa, increasing its hindering effect and thus decreasing or avoiding the penetration of contaminants and biocides. Results Several compositions have been formulated and assayed; the hydrophobic formulation F1, the hydrophilic formulation F2 and the liposomal formulations F3 with lipids and structure similar to those present in cutaneous keratinous tissues. These all induced a waterproofing effect with the liposomal formulation F3 and in particular the hydrophobic formulation F1 inducing the greatest decrease in Trans-mucosal water loss. Permeation of the three biocides Fungitrol, Propiconazole and Permethrin was evaluated in skin and in untreated and treated (F1, F2, F3) oral mucosal membranes. The influence of the three formulations on each of the biocides used is considerable, reaching values significantly equal to those of the skin for formulation 1. While hydrophilic formulation 2 greatly increases permeability, hydrophobic formulation 1 and liposomal formulation 3 reduce permeability, in approximately 90% and 60% respectively, providing an almost skin-like barrier effect. Conclusions Therefore, this work presents two formulations that induce mucosal impermeability by reducing the penetration of three model biocides.