Background: Fungal keratitis (FK), a major cause of blindness, remains challenging to treat due to poor drug penetration and antifungal resistance. Amphotericin-B (AmB), a water-insoluble and low-permeability, necessitates innovative delivery systems to improve its therapeutic efficacy. Methods: AmB was encapsulated within oleosomes (Ole) prepared using the ethanol injection method, using phosphatidylcholine (Lipoid S100) and sodium oleate, resulting in nanosized spherical globules. The optimized Ole were characterized, then the selected Ole were incorporated into sodium polyacrylate/PEG/chitosan-based microneedles (AmB-Ole/MNs) to improve ocular delivery by creating transient microchannels on the eye surface. Results: The optimized Ole showed a droplet size of (175 ± 0.78 nm), polydispersity index of (0.33 ± 0.04), zeta potential of (31 ± 0.43 mV), high entrapment efficiency (91±0.63%), and improved stability, bioavailability, and controlled drug release. The AmB-Ole/ MNs system increases corneal penetration and contact time via transient microchannels in the eye surface, achieving sustained drug delivery over 72 hours with 70% ex vivo permeation over 80 hours compared to AmB. In vitro antifungal activity and histopatho logical examination showed that the AmB-Ole/MNs system has potent biofilm disruption (>90%) and 27 mm and 32 mm zones of inhibition against Candida albicans and Aspergillus niger, respectively. The Cytotoxicity test showed reduced AmB toxicity with biocompatibility and in vivo rabbit model, ocular tolerance by targeting TLR4/NLRP3 pathways and histopathological studies. Conclusion: The AmB-Ole/MNs system as an innovative ocular delivery platform for fungal keratitis offers sustained drug release, enhanced permeation, potent antifungal activity, and reduced toxicity. AmB-Ole/MNs showed promise for ocular AmB delivery for FK.