Fungal keratitis (FK), the major cause of ocular morbidity worldwide, is commonly treated with the amphotericin B (AmB) eye drops extemporaneously prepared from marketed parenteral formulations. However, these in-house prepared AmB eye drops have the drawbacks of poor ocular bioavailability and eye irritation. The aim of this study was to develop AmB-loaded fibroin nanoparticles (AmB-FNPs), in combination with the polymer PEG 400, as ready-to-use eye drops for FK. The AmB-FNPs were prepared by desolvation method. All AmB-FNPs exhibited homogeneous spherical particles with a mean size of ~ 270 nm, the zeta potential of ~ - 17 mV, and an entrapment efficiency of ~ 65%. Using X-ray diffraction and UV analysis, AmB demonstrated amorphous molecular dispersion and monomeric form when entrapped in the FNPs. Interestingly, in dissolution studies, although AmB-FNPs showed no detectable drug release in sink condition, they still possessed good antifungal activity against Candida albicans. Potentially, AmB-FNPs showed less cytotoxicity in human corneal epithelial cell line compared to the marketed AmB deoxycholate.