<p>Chlorogenic acid (CA), a principal bioactive constituent prevalent in numerous Chinese herbal medicines, exhibits well-documented antibacterial efficacy. However, CA is rapidly metabolized in the intestine and liver, and the specific mechanisms of its metabolites against <i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>) biofilms remain unclear. This study demonstrated that shikimic acid, a key metabolite of CA, effectively inhibits biofilm formation in clinical <i>P. aeruginosa</i> strains without impeding bacterial growth. This antibiofilm activity was linked to the significant suppression of extracellular polymeric substance (EPS) and alginate production, as well as the impairment of bacterial motility. By integrating transcriptome analysis with RT-PCR validation, it is postulated that the antibiofilm action of shikimic acid may be attributed to its regulatory effects on the constituents of the EPS within the biofilm, especially extracellular polysaccharides. Moreover, shikimic acid predominantly downregulated genes associated with alginate synthesis, among which <i>algD</i> exhibited the most significant expression alteration. In order to ascertain the role of <i>algD</i> in the antibiofilm action of shikimic acid, an <i>algD</i> knockout strain, PA2209Δ<i>algD</i>, was constructed. The results demonstrated that shikimic acid did not exerted notable inhibitory effects on biofilm formation or alginate production in the <i>algD</i> knockout strains, suggesting that the <i>algD</i> gene is essential for the antibiofilm effect of shikimic acid. These findings elucidate a novel mechanism by which a CA-derived metabolite disrupts biofilm integrity by targeting the <i>algD</i>-dependent alginate biosynthesis pathway, positioning shikimic acid as a potential lead for developing non-bactericidal anti-biofilm agents.</p>