Background: Alginate is one of the main extracellular polymeric substances (EPS) in biofilms of Cystic Fibrosis (CF) patients suffering from pulmonary infections. Gentamicin sulfate (GS) can strongly bind to alginate resulting in loss of pharmacological activity; however neither the mechanism nor its repercussion is fully understood. In this study, we investigated how GS modifies the alginate macromolecular network and its microenvironment. Material and methods: Alginate gels of two different compositions (either enriched in guluronate units (G) or enriched in mannuronate units (M)) were crosslinked with Ca2+ and exposed to GS at varying times and concentrations. The complexes formed were characterized via turbidimetry, mechanical tests, swelling assay, calorimetry techniques, nuclear magnetic resonance, Ca2+ displacement, macromolecular probe diffusion and pH alteration. Results: In presence of GS, the alginate network and its environment undergo a tremendous reorganization in terms of gel density, stiffness, diffusion property, presence and state of the water molecules. We noted that the intensity of those alterations is directly dependent on the polysaccharide motif composition (ratio M/G). Conclusion: Our results underline the importance of alginate as biofilm component, its pernicious role during antibiotherapy and could represent a potential macromolecular target to improve anti-infectious therapies.