Eclipta prostrata (L.) L. belonging to the Astearaceae family has been long used worldwide by various communities as a traditional medicine. We studied the antibacterial activity of different extracts from Eclipta prostrata (L.) L. against Aeromonas hydrophila, Aeromonas dhakensis, and Vibrio parahaemolyticus . The processing of this extract using repeated column and thin layer chromatographic techniques resulted in the isolation of three compounds including luteolin (1), kaempferol (2), and kaempferol-3-O-α-L-rhamnopyranoside (3), which were identified by NMR. Among them, kaempfreol-3-O-α-L-rhamnopyranoside was isolated for the first time and shows good antibacterial activity against Aeromonas hydrophila which is major pathogen for the aquaculture industry. The results demonstrate the beneficial effects of E. prostrata as an antimicrobial and bioactive compound for medicinal usages.