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Poly-(lactic-<i>co</i>-glycolic) acid (PLGA) is a biodegradable, biosafe, and biocompatible copolymer. The <i>Aspergillus</i> section <i>Nigri</i> causes otomycosis localized in the external auditory canal. In this research, <i>Aspergillus brasiliensis</i>, a species belonging to the <i>Nigri</i> section, was tested. Coumarin 6 and pterostilbene loaded in poly-(lactic-<i>co</i>-glycolic) acid nanoparticles (PLGA-coumarin6-NPs and PLGA-PTB-NPs) were tested for fungal cell uptake and antifungal ability against <i>A. brasiliensis</i> biofilm, respectively. Moreover, the activity of PLGA-PTB-NPs in inhibiting the <i>A. brasiliensis</i> infection was tested using <i>Galleria mellonella</i> larvae. The results showed a fluorescence signal, after 50 nm PLGA-coumarin6-NPs treatment, inside <i>A. brasiliensis</i> hyphae and along the entire thickness of the biofilm matrix, which was indicative of an efficient NP uptake. Regarding antifungal activity, a reduction in <i>A. brasiliensis</i> biofilm formation and mature biofilm with PLGA-PTB-NPs has been demonstrated. Moreover, in vivo experiments showed a significant reduction in mortality of infected larvae after injection of PLGA-PTB-NPs compared to free PTB at the same concentration. In conclusion, the PLGA-NPs system can increase the bioavailability of PTB in <i>Aspergillus</i> section <i>Nigri</i> biofilm by overcoming the biofilm matrix barrier and delivering PTB to fungal cells.