Eugenol exhibits antibacterial activity against several bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, etc. Also eugenols and its derivatives are known to reduce the biofilm at sub MIC levels. In the current study we evaluated the effect of sub MIC levels of isoeugenol on biofilm formation against Aeromonas hydrophila strain. Biofilm forming capability was reduced in a concentration dependent manner at all tested subMIC values of isoeugenol. Additionally, β-galactosidase assay suggests isoeugenol down regulates the expression of putative type IV pili operon pilMNOPQ genes. Similarly reduced biofilm formation phenotype was also observed in PilMNOPQ operon mutant strain. Thus the study suggests that the phenotype of PilMNOPQ mutant strain of reduced biofilm forming ability appears comparable to that of use of isoeugenol to inhibit biofilm formation of Aeromonas hydrophila.