Objective To explore the potential mechanism of carvacrol on inhibiting the formation of biofilm of hypervirulent Klebsiella pneumoniae (hvKP). Methods The possible mechanisms of carvacrol were analyzed based on the detection of its effects on the formation and morphology of biofilms, changes in extracellular polysaccharide and capsule polysaccharide content, as well as changes in the expression levels of biofilm-related genes rmpA2, magA, mrkA, mrkB, and treC of hvKP. Results The minimum inhibitory concentration of carvacrol on hvKP was 512 μg/mL, with an obvious inhibitory effect on the biofilm formation of hvKP, presenting a concentration-depen-dent effect. Under the scanning electron microscope, it was observed that the biofilm structure was loose and the intercellular connections were not dense under the intervention of carvacrol. The Congo Red adsorption test and m-hydroxybiphenyl colorimetric method showed that carvacrol could reduce the content of capsule polysaccharides of hvKP, but didn't affect the total extracellular polysaccharide content. Fluorescence quantitative polymerase chain reaction (PCR) showed that under the effect of carvacrol at sub-inhibitory concentration, the synthesis of capsule polysaccharide, expression levels of sugar transport system and pili adhesion-related genes all decreased by more than 50%. Conclusion Carvacrol has a significant inhibitory effect on the formation of biofilm in hvKP, and its mechanism may be related to the decrease of synthesis of capsule polysaccharide as well as expression of biofilm-related genes, such as sugar transport system and pili adhesion.