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Objective: Considering the emergence of biofilm-associated pathogens with multidrug resistance, the objective(s) of the present study was to evaluate the anti-virulence property of Syzygium aromaticum extracts/fractions against 2 multidrug-resistant catheter isolates. Materials and Methods: Pulverized clove buds were subjected to bioactivity-guided isolation to identify the bioactive extract/fraction(s) with significant anti-virulence property. The clove bud powder was subjected to Soxhlet extraction and all these extracts were investigated in terms of anti-virulent efficacy using initial readout assays. Bioassay-guided partial purification was performed through column and size exclusion chromatography. Detailed testing of the anti-virulence and anti-biofilm activity of sub-minimum inhibitory concentration (sub-MIC) levels of the active fraction, was performed besides the cytotoxicity profiling in RAW 264.7 cells. Results: Gas chromatography-mass spectrometry (GC-MS) analysis of the clove bioactive fraction-2 (CBF-2), done after the bioassay-guided fractionation, revealed eugenol as the major bioactive ingredient present in CBF-2. Reduced mRNA levels of virulence factor genes after CBF-2 (700 μg/ml) treatment correlated well with the respective phenotypic assays (p<0.001). Similarly, CBF-2 (700 μg/ml) treatment exhibited significantly low mRNA levels of quorum sensing (QS) receptor genes compared to their cognate synthase genes (p<0.001). Crystal violet staining and scanning electron micrographs of CBF-2-treated biofilms showed lesser macrocolonies with remarkably simplified architecture. Cytotoxic evaluation of CBF-2 suggested a minute reduction in viability even at the highest tested concentration (1600 μg/ml, p<0.05). Conclusion: The present study indicated that eugenol-rich CBF-2 has potent anti-virulence and anti-biofilm activity against urinary catheter isolates and can be regarded as an alternative for treatment of catheter-associated urinary tract infections. [ABSTRACT FROM AUTHOR]