BACKGROUND A biofilm is an extracellular polymeric substance (EPS) composed of polysaccharides, proteins, nucleic acids, and lipids that impede antibiotics and immune cells, thus providing a shielded environment for bacterial growth. Due to biofilm formation, some microbes can show up to 1000 fold increased resistance towards the antibiotics than the normal planktonic forms. The study was conducted to screen the crude extracts of medicinal plants used in Nepal for their in vitro antibiofilm activities. METHODS Total phenolic and total flavonoid contents were determined by using a Folin-Ciocalteau reagent and aluminium trichloride method, respectively. Resazurin assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The initial antibiofilm activities and their inhibitory concentration (IC50) values were determined by the microtiter based modified crystal violet staining method. RESULTS Out of 25 different plant extracts were used for the study, methanolic extracts of 20 plants showed a biofilm inhibition activity against five different strong biofilm producing Escherichia coli strains. Calotropis gigantea exhibited inhibition against all five different E. coli strains with IC50 values ranging from 299.7 ± 20.5 to 427.4 ± 2.7 μg/mL. Apart from that, Eclipta prostrata also showed biofilm formation inhibition, followed by Eupatorium adenophorum, Moringa oleifera, Ocimum tenuifolium, Oxalis lantifolia, Prunus persica, and Urtica parviflora. The extracts of C. gigantea, E. prostrata, Mangifera indica, O. tenuifolium, P. persica, and U. parviflora exhibited a moderate to poor MIC value ranging from 625 to 2500 μg/mL. The highest amount of phenolic content (TPC) was found in Acacia catechu followed by Morus alba, which was 38.9 and 25.1 mg gallic acid equivalents, respectively. The highest amount of flavonoid content was found in A. catechu followed by M. indica, which was 27.1 and 20.8 mg quercetin equivalents, respectively. CONCLUSIONS Extracts of C. gigantea, E. prostrata, P. persica, U. parviflora, and O. tenuifolium showed antibacterial as well as antibiofilm activity against pathogenic and strong biofilm producing E. coli. Thus, extracts or the pure compound from these medicinal plants could be used as antibiotics in the future.