Stenotrophomonas maltophilia is a gram-negative opportunistic pathogenic bacterium that is associated with hospital- and community-acquired infections. Ithas a set of virulence factors, such as biofilm formation and extracellular enzymes, that are mostly regulated via quorum sensing (QS) systems. Azithromycin (AZM) is a macrolide that is well known for its anti-virulence effects, including anti-QS and antibiofilm effects. Additionally, some spice essential oils have been reported to inhibit bacterial virulence. This study evaluated the effect of AZM and Fenugreek Oil (FO), a spice essential oil from Fenugreek seeds, against some virulence factors of multidrug-resistant Stenotrophomonas maltophilia clinical isolates. Both AZM and FO showed significant inhibitory effects against protease activity, where all tested isolates showed 100% loss of the halo zone formed in skimmed milk agar test with AZM and a 25 to 35% reduction in the zone with FO. A mean reduction in the interstitial surface area of 34.4% and 35.5% was detected with AZM and FO, respectively, in the twitching motility assay. While AZM showed a significant effect in reducing biofilm formation by S. maltophilia isolates (mean inhibition of 49.7%), the reducing effect of FO (18.5%) was not significant. Genotypically, exposure of S. maltophilia clinical isolates to AZM and FO significantly reduced the expression of protease-encoding genes (stmPr1, stmPr2 and StmPr3) and a quorum sensing gene (rpfC).