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Biofilms are defined as flexible films prepared from biological materials such as proteins or polysaccharides, which act as barriers to external elements and can consequently protect the products and increase their shelf life. The objective of this study was to evaluate the effects of hydrophilic and hydrophobic plasticizers and mixtures thereof in the characterization of alginate biofilms. Films plasticized with glycerol were less hygroscopic than films plasticized with TC (tributyl citrate) or with TC/glycerol mixtures. The presence of TC in the plasticization of the films turn them more hygroscopic, thus confirming their excellent performance in protecting against dissolution and in water vapor permeability. Films plasticized with pure TC were the least water?soluble and showed the higher tensile stress, having a globular microstructure, which was attenuated as the TC was partially substituted by glycerol, making the structure more compact but not as homogenous as films containing only glycerol as the plasticizer. Films plasticized with pure TC and with TC/glycerol mixtures were opaque and showed only one glass transition temperature (Tg). © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 48263.