Abstract The purpose of the present research was to formulate ocular inserts of norfloxacin along with bioadhesive polymers to sustain drug release and assess its antimicrobial potential against E. coli, in vitro. Ocular inserts of norfloxacin were prepared by film casting method using bioadhesive polymers HPMC/ SCMC/ sodium alginate/ chitosan/carbopol 934 and PVP K30 as film forming polymer. The formulated bioadhesive norfloxacin ocular inserts were screened for various pharmacotechnical parameters and in vitro drug release. The optimized formulation was subjected to HET CAM test and antimicrobial efficacy against E. coli. Of the screened formulations, maximum bioadhesive strength (36.5 ± 0.10 x 10-3 dynes/cm2) was observed for chitosan based ocular insert. Kinetic modeling of the in vitro drug release data revealed matrix based release was governed by both diffusion and swelling phenomena. Ocular insert consisting formulated with chitosan was found promising, as it showed acceptable pH, good bioadhesive strength, sustained drug release (98.33% at 6 h) and active against selected microorganism.