Invasive aspergillosis is a serious threat to immunodeficient and critically ill patients caused mainly by the fungus Aspergillus fumigatus. Here, poly(glycidol)-based nanogels (NGs) are proposed as delivery vehicles for antifungal agents for sustained drug release. NGs are formed by simple self-assembly of random copolymers, followed by oxidative cross-linking of thiol functionalities. We investigate the impact of copolymer amphiphilicity on NG interaction with mature fungal hyphae in order to select the optimal drug delivery system for model antifungal drug amphotericin B. The results show that drug-loaded NGs decrease minimal inhibitory concentration (MIC) for around four times and slow down the fungal biofilm synthesis at concentrations lower than MIC. Our results suggest that amphiphilicity of nanoparticle's polymer matrix is an important factor in understanding the action of nanocarriers toward fungal cells and should be considered in the development of nanoparticle-based antifungal therapy.