The use of indwelling medical devices (e.g. pacemakers, prosthetic joints, catheters, etc) continues to increase, yet these devices are all too often complicated by infections with biofilm-forming microbes with increased resistance to antimicrobial agents and host defense mechanisms. We investigated the ability of chitosan, a polymer isolated from crustacean exoskeletons, to damage biofilms formed by the pathogenic fungus Cryptococcus neoformans. Using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay and CFU determinations, we showed that chitosan significantly reduced both the metabolic activity of the biofilms and cell viability, respectively. We further demonstrated that chitosan penetrated biofilms and damaged fungal cells using confocal and scanning electron microscopy. Notably, melanization, an important virulence determinant of C. neoformans, did not protect cryptococcal biofilms against chitosan. The chitosan concentrations used in this study to evaluate fungal biofilm susceptibility were not toxic to human endothelial cells. Our results indicate that cryptococcal biofilms are susceptible to treatment with chitosan, suggesting an option for the prevention or treatment of fungal biofilms on indwelling medical devices.