Abstract: In the present study, the antibacterial activity of postbiotics PostbioYDFF against Pseudomonas aeruginosa (P. aeruginosa) and its mechanism of action were examined by analyzing its effects on biofilm formation and changes in the ultrastructure and transcriptome. Meanwhile, to validate transcriptome data, qPCR was conducted. The results showed that PostbioYDFF exhibited significant bacteriostatic activity against P. aeruginosa with a diameter of inhibition zone growth of 28.9±0.3 mm and the MIC value of 100 μL/mL. In addition, compared with the control group, PostbioYDFF was able to significantly inhibit biofilm formation by 64.96% (P<0.01) and disrupted established biofilms by 30.66% (P<0.05). It was also observed that PostbioYDFF disrupted the bacterial structure and attenuated bacterial motility (swimming, swarming and twitching). Through RNA-seq analysis combined with qPCR validation, a total of 503 significantly different genes were found after PostbioYDFF treatment as compared with the control group, including 237 significantly up-regulated genes which were mainly enriched in ribosomal, RNA degradation, oxidative phosphorylation and other pathways, and 266 significantly down-regulated genes which were mainly enriched in the energy metabolism, lipid metabolism, and two-component system and other pathways. This study provides an important theoretical basis for the prevention and control of P. aeruginosa by PostbioYDFF, and points out a potential application in food preservation.