The escalating threat of microbial resistance underscores the urgent need for innovative solutions, including natural agents capable of attenuating virulence. This study explores the antimicrobial and anti-virulence potential of essential oils (EOs) derived from Sphagneticola trilobata against Pseudomonas aeruginosa. Through GC/MS analysis, volatile metabolites from the flower heads and leaves/stems of Egyptian S. trilobata were identified, revealing 43 and 62 components, respectively. Key compounds included α-phellandrene, α-pinene, D-limonene, and α-thujene. The Minimum Inhibitory Concentrations (MIC) of flower head and leaf/stem EOs against P. aeruginosa were 1.17% and 1.75% v/v, respectively. At sub-MIC doses (1/8th of the MIC), the EOs exhibited significant anti-virulence properties, including complete inhibition of protease activity and disruption of biofilm formation, which are crucial for bacterial survival and pathogenicity. Additionally, they effectively suppressed the expression of quorum sensing genes, which are essential for bacterial communication and virulence. Virtual screening of four major EO components (+)-(R)-limonene, (±)-α-pinene, α-phellandrene, and α-thujene against five critical protein targets involved in biofilm formation, quorum sensing, virulence, and protease activity in P. aeruginosa further supported their anti-virulence and antibiofilm actions, showing high affinity for these targets. These findings suggest that the EOs of S. trilobata hold great potential as natural virulence attenuating agents, particularly against biofilm-forming pathogens like P. aeruginosa.