This study investigates the antibacterial and anti-biofilm activities of three high-molecular-mass metabolites (biosurfactant, lipoteichoic acid, and exopolysaccharide) from Lacticaseibacillus rhamnosus YT. Additionally, the bacteriostatic effects of these three metabolites on different strains isolated from raw milk were examined. The results showed that based on inhibition zone and growth curve assays, these metabolites exhibited varying degrees of antibacterial activity against six common food spoilage microorganisms at concentrations of 50 and 100 mg/mL. The minimum inhibitory concentration (MIC) against Salmonella enterica ranged from 60 to 100 mg/mL, while those against Pseudomonas aeruginosa ranged from 80 to 150 mg/mL. At 1/4 MIC concentration, the biosurfactant inhibited the biofilms of S. enterica and P. aeruginosa by 43.33% and 36.26%, respectively, compared with 29.72% and 24.19% for lipoteichoic acid. The anti-biofilm capacity of the exopolysaccharide was significantly weaker than those of the biosurfactant and lipoteichoic acid. When the concentration increased to the MIC, the disruption rates of mature biofilms by the three metabolites significantly increased. Eight strains from raw milk were isolated and identified by 16S rDNA sequencing. The diameters of the inhibition zones of the biosurfactant against these strains ranged from 9.00 to 14.00 mm, while those of the lipoteichoic acid ranged from 9.00 to 10.00 mm. At 120 mg/mL concentration, the exopolysaccharide showed no significant antibacterial effects. These findings support the development of natural antibacterial agents based on the metabolites of L. rhamnosus YT, thereby reducing the risk of food contamination and meeting consumer demand for green and safe food products.