Limonia acidissima is a medicinal plant commonly used for multitude of ailments. In this context, the validation of traditionally used medicinal plant that the fruit extracts of L. acidissima exhibit antibiofilm activity against the predominant fish pathogen Aeromonas hydrophila was assessed. Among ten isolates of Aeromonas spp, KUAH1 showed strong biofilm formation and was studied further. The methanol (LA-M) and ethyl acetate (LA-EA) fraction of Limonia fruit extract clearly demonstrated significant (p ≤ 0.005) antibiofilm activity of 58 to 94% and 54 to 77% respectively. Furthermore, the potential of Limonia fruit extracts against some of the biofilm associated factors were tested by swimming and swarming motility assay, XTT and anti-haemolytic activity assay. Extracellular protein analysis revealed differential protein expression at molecular weight corresponding to 30-60 kDa. This is the first report on antibiofilm activity of L. acidissima fruit extracts, signifying the scope for development of complementary medicine to treat Aeromonas biofilm-associated infections.