Novel drug delivery systems have emerged to treat bacterial keratitis, an acute infection of the cornea. In this study, besifloxacin HCl loaded insert formulations were designed and investigated in vitro, ex vivo and in vivo for the treatment of bacterial keratitis. Besifloxacin HCl (BH) or BH-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) complex containing poly(caprolactone)/polyethylene glycol (PLC/PEG) fibrous inserts were prepared with an electrospinning method. These fibrous inserts were coated with mucoadhesive polymers such as sodium alginate (SA) or thiolated sodium alginate (TSA). Developed inserts compared to commercially available drug and it was found that coating of the insert surfaces with SA and TSA, increases bioadhesion of the formulations. Insert formulations showed a burst release in the first 2 days followed by a slow-release profile. Ex vivo transport studies showed that HP-beta-CD possessed a drug delivery level close to the commercial drug. Both TSA coated inserts as well as inserts containing HP-beta-CD-drug complex were effectively reducing bacterial keratitis in rabbit eyes upon single-dose application compared to multiple dosing with the commercial drug. Consequently, TSA coated inserts as well as the inserts containing HP-beta-CD-drug complex, may be potential alternatives to conventional market product by reducing the application frequency in the clinic leading to increased patient compliance.