An aquatic ethno-medicinal herb, Monochoria hastata (L.) Solms belonging to the family Pontederiaceae is used in Indian sub-continent as remedy of several ailments like wounds and boils, gastropathy, hepatopathy etc The aim of the present work was to assess the antibiofilm activity of ethyl acetate extract of the aerial part of this plant.. TLC and LC-MS was performed to visualize the presence of active principles with their mass profile and Antibiofilm activities of the purified compound were assessed following the standard biofilm production assay protocol (Djordjevic et al., 2002) against the Gram-positive strain, Staphylococcus epidermidis MTCC 3086 (S. epidermidis) and Gram negative strain, Vibrio cholerae MTCC 3906 (V. cholerae). The extracted compounds were centralized in a single spot (S1) at retardation factor (Rf) value of 0.92 in TLC and peaks with their characteristic features and relative abundances were detected at different retention times in LC-MS and exhibited selective antibiofilm activities without any cytotoxic effect in human cell line.