Background <p>The urgent need for novel antimicrobial agents arises from the escalating threat of antibiotic-resistant pathogens. Tackling the resistance mechanisms through the application of resistance modifying agents in combination to antimicrobials represents an effective strategy to combat antimicrobial resistance and to enhance the antimicrobial activity. Screening of plant extracts, essential oils and their active compounds for potential resistance modifying properties has proven effective on both a small and large scale. In this study, we define the metabolomic profile of essential oils extracted from different parts of <i>Cymbopogon citratus</i> (DC.) Stapf (lemongrass), and characterize their antioxidant and virulence attenuating activities.</p> Methods <p>The major active ingredients of essential oils extracted from different parts of <i>Cymbopogon citratus</i> (lemongrass) were identified by GC-MS analysis. The minimum inhibitory concentration (MIC) of the essential oils against <i>P. aeruginosa</i> PAO1, <i>Staphylococcus aureus</i> ATCC 6538, and <i>Candida albicans</i> ATCC 10,261 was determined using the broth microdilution assay. Antibiofilm and antiprotease activities were phenotypically evaluated for the extracted lemongrass essential oils for the same standard strains and clinical isolates for the same microbes. Also, the effects on microbial virulence were validated by RT-qPCR against a subset of genes regulating biofilm, quorum sensing, and stress response in <i>P. aeruginosa</i>, and <i>S. aureus</i>. Furthermore, the antioxidant activities of the different essential oils extracted were evaluated using DPPH, β-carotene/linoleic acid and FRAP methods.</p> Results <p>GC-MS analysis of the essential oils of <i>Cymbopogon citratus</i> revealed the presence of 47 different compounds distributed in the different plant organs. Active constituents such as geranial, neral, myrcene, nerolic acid, linalool, iso-citral, trans iso-citral, and neryl acetate were more abundant in the essential oil extracted from leaves (89.77%), followed by stems (82.92%) and finally roots (52.02%). <i>S. aureus</i>, and <i>C. albicans</i> were more sensitive to <i>C. citratus</i> essential oils than Gram negative <i>P. aeruginosa</i>. Incorporation of sub-MIC doses of essential oils into the culture media was sufficient to disrupt the formation of microbial biofilms in <i>P. aeruginosa</i>, <i>S. aureus</i>, and <i>C. albicans</i>, and to inactivate the proteolytic activities of microbial proteases. The essential oils of <i>C. citratus</i> showed promising and broad-spectrum biofilm eradicating activity. Sub-MIC doses of lemongrass oil dramatically reduced the expression of <i>relA</i>, <i>pslA</i>, and <i>spoT</i> in <i>P. aeruginosa</i>, and <i>agrA</i>, <i>icaA</i>, and <i>sigB</i> in <i>S. aureus</i> indicating a broad-spectrum anti-virulence activity.</p> Conclusion <p>Our study identifies <i>C. citratus</i> essential oils as potential virulence-attenuating agents. Essential oils extracted from the leaves, stems, and roots of <i>C. citratus</i> exhibited significant antimicrobial, anti-biofilm, anti-virulence, and antioxidant activities, effectively combating microbial resistance. These findings suggest that <i>C. citratus</i> essential oils could be a valuable natural alternative in the fight against resistant pathogens.</p>