Pseudomonas aeruginosa is a conditional Gram-negative pathogen that produces extracellular virulence factors that can lead to bloodstream invasion, severely harm tissues, and disseminate bacteria, ultimately leading to various diseases. In this study, lactic acid bacteria (LAB) with strong antagonistic ability against P. aeruginosa were screened, and the regulatory mechanism of LAB against P. aeruginosa was evaluated. The results showed that the three selected LAB strains had strong inhibition ability on the growth, biofilm formation, and pyocyanin expression of P. aeruginosa and a promoting effect on the expression of autoinducer-2. Among them, Lactipantibacillus plantarum (Lp. plantarum) LPyang is capable of affecting the metabolic processes of P. aeruginosa by influencing metabolic substances, such as LysoPC, oxidized glutathione, betaine, etc. These results indicate that LPyang reduces the infectivity of P. aeruginosa through inhibition of its growth, biofilm formation, pyocyanin expression, and regulation of its metabolome. This study provides new insights into the antagonistic activity of Lp. plantarum LPyang against P. aeruginosa.