Background/Objectives: Keratitis is an ocular disease caused by microbial infection or by non-infectious damage due to UV light exposure, chemical exposure, or eye injuries. Methods: Moxifloxacin-loaded ufasomes (MOX-UFAs) were optimized using a full factorial design (1².2³) after being prepared by the vortex mixing method. The study evaluated the effects of the oleic acid amount, surface active agent (SAA) amount, and SAA type as independent factors on the entrapment efficiency percent (EE%), particle size (PS), polydispersity index (PDI), zeta potential (ZP), and the amount released after 6 h (Q6h%). Results: The optimized ufasomes (UFAs) formulation was spherical, with an EE% of 78.37 ± 3.91%, PS of 203.13 ± 20.31 nm, PDI of 0.334 ± 0.016, and ZP of −25.42 ± 1.27 mV. The in vitro release of moxifloxacin (MOX) from the UFAs was maintained for more than 6 h in the range of 40.0–75.0%. The optimum MOX-UFAs formulation was incorporated into an in situ gel (Pluronic F-127/HPMC K4M). The ex vivo studies (corneal permeation and confocal laser scanning microscopy) proved the successful retention of the MOX-UFAs-laden in situ gel. Furthermore, the in vitro and in vivo antimicrobial studies revealed their significant antimicrobial effect against Pseudomonas aeruginosa. In addition, the Draize test proved the tolerability of MOX-UFAs-laden in situ gel in animals. Conclusions: The incorporation of MOX-UFAs into Pluronic F-127/HPMC K4M in situ gel could successfully provide sustained ocular delivery and improve the bioavailability of MOX for the management of keratitis.