Lactic acid bacteria are commonly present in various sources and possess significant probiotic properties. They can inhibit pathogenic bacteria and fungi simultaneously, making them promising candidates as bio-preservatives. This study investigated two potential probiotic strains: Lactiplantibacillus plantarum LR5-2 (isolated from fermented meat products) and Lacticaseibacillus rhamnosus SQ63 (isolated from infant feces). The study evaluated their aggregation ability, anti-pathogenic activity, safety, and tolerance to gastrointestinal conditions, phenol, and bile salts. Additionally, their biological control potential against Penicillium expansum on fresh grapes was assessed. The results demonstrated that both strains exhibited high survival rates under extreme gastrointestinal conditions, enhanced Auto-aggregation, co-aggregation, and hydrophobicity. They displayed strong antioxidant activity and significant antibacterial effects against 11 pathogenic fungi and foodborne pathogens. Biosafety testing revealed that both strains are sensitive to most antibiotics, do not produce biogenic amines, and exhibit no hemolytic or DNase activity. In grapes, L. plantarum LR5-2 and L. rhamnosus SQ63 significantly reduced the incidence and disease index of P. expansum infection. In conclusion, the characterization analysis and bio-preservation experiments revealed that LR5-2 and SQ63 have strong potential as probiotics and bio-preservatives.