The dissemination of antibiotic resistance genes (ARGs) via conjugative plasmids is a major driver of the antimicrobial resistance crisis. The optrA gene, which confers cross-resistance to linezolid and florfenicol, is frequently detected in Enterococcus isolates and disseminated via plasmids. This study investigated the inhibitory effect of carvacrol on horizontal transfer of an optrA-carrying plasmid in porcine Enterococcus faecalis. In vitro conjugation assays showed that subinhibitory carvacrol (1/32 MIC, 16 µg/mL) significantly reduced conjugation efficiency in a dose- and time-dependent manner. In vivo (mouse model) treatment with 32 μg/mL carvacrol decreased transconjugant recovery by 62.8%. Mechanistically, carvacrol disrupted membrane integrity, depleted intracellular ATP, sup-pressed ROS production, reduced biofilm formation, and downregulated conjugation-associated genes (type VI secretion protein, conjugal transfer protein, prgB, asa1) as shown by transcriptomics and RT-qPCR. Collectively, carvacrol inhibits optrA plasmid transfer via multi-target mechanisms involving membrane damage, energy metabolism interference, and suppression of conjugation gene expression. This study highlights carvacrol as a natural agent to control the transfer of ARGs.