PURPOSE: Chronic infections of Candida albicans are characterised by the embedding of budding and entwined filamentous fungal cells into biofilms. The biofilms are refractory to many drugs and Candida biofilms are associated with ocular fungal infections. The objective was to test the activity of nanoparticulate amphotericin B (AmB) against Candida biofilms. METHODS: AmB was encapsulated in the Molecular Envelope Technology (MET, N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan) nanoparticles and tested against Candida biofilms in vitro. Confocal laser scanning microscopy (CLSM) imaging of MET nanoparticles’ penetration into experimental biofilms was carried out and a MET-AmB eye drop formulation was tested for its stability. RESULTS: MET-AmB formulations demonstrated superior activity towards C. albicans biofilms in vitro with the EC50 being ~30 times lower than AmB alone (EC50 MET-AmB = 1.176 µg mL−1, EC50 AmB alone = 29.09 µg mL−1 ). A similar superior activity was found for Candida glabrata biofilms, where the EC50 was ~10× lower than AmB alone (EC50 MET-AmB = 0.0253 µg mL−1, EC50 AmB alone = 0.289 µg mL−1 ). CLSM imaging revealed that MET nanoparticles penetrated through the C. albicans biofilm matrix and bound to fungal cells. The activity of MET-AmB was no different from the activity of AmB alone against C. albicans cells in suspension (MET-AmB MIC90 = 0.125 µg mL−1, AmB alone MIC90 = 0.250 µg mL−1 ). MET-AmB eye drops were stable at room temperature for at least 28 days. CONCLUSIONS: These biofilm activity findings raise the possibility that MET-loaded nanoparti-cles may be used to tackle Candida biofilm infections, such as refractory ocular fungal infections.