Bacterial infections have increased dramatically due to microbial biofilm formation resulting in chronic pathological conditions in human subjects. Microbial biofilm causes poor drug penetration and antibiotic resistance, which has made site-specific sustained drug delivery the most appropriate option. Our work entails fabrication of ciprofloxacin hydrochloride (CPX) loaded nanofibers using polyvinyl alcohol (PVA) and poly(meth) methacrylate (PMMA) employing electrospinning method to form PVA:PMMA:CPX nanofibers mat. These nanofibers mat were optimized, characterized, and further subjected to anti-biofilm activity. Microscopic images revealed average nanofibers diameter of 243 +/- 80 nm with smooth surface morphology. Analytical graphs and thermal analysis confirmed drug encapsulation and drug-polymer compatibility. In vitro studies demonstrated sustained release of CPX for 22 days displaying Hixon Crowell and two-stage desorption kinetics. Anti-biofilm activity showed zones of inhibition 3.0 +/- 0.5 cm, 2.8 +/- 0.1 cm, 2.9 +/- 0.2 cm for Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, respectively which was well above the minimum inhibitory concentration levels of the bacteria forming biofilm. Conclusively, these nanofibers mat have potential to be used as an anti-biofilm product.