Lactiplantibacillus pentosus SM1 displayed notable resistance to acidic environments, with over 76% survival after 3 h in low pH conditions, and demonstrated viability in simulated gastrointestinal fluids. The Lpb. pentosus SM1 exhibited surface hydrophobicity, auto-aggregation, co-aggregation, and adhesion values of 59.60%, 38.80%, 44.90%, and 13.10% respectively. The anti-adhesion capability of Lpb. pentosus SM1 against Listeria monocytogenes adhesion to Caco-2 cells was demonstrated by its ability to compete with, inhibit, and displace at rates of 54.10%, 48.80%, and 23.90%, respectively. The Lpb. pentosus SM1 exhibited broad-spectrum anti-microbial activity, particularly against Gram-positive bacteria, and inhibited biofilm formation. The strain also showcased antioxidant activity, effectively scavenging DPPH (57.60%) and ABTS (60.54%) radicals. Notably, it absorbed 51.60% of cholesterol, highlighting its potential for cholesterol-lowering benefits. The cytotoxicity values were measured at 36.57 mg/mL for HT-29 and 38.20 mg/mL for Hela cancer cell lines. Importantly, Lpb. pentosus SM1 was free of hemolytic activity and biogenic amines, demonstrating a favorable safety profile. Lpb. pentosus SM1 exhibited the strongest resistance to ampicillin, while its resistance to erythromycin was the weakest, with inhibition zones measuring 15.50 mm and 26.40 mm, respectively. These findings suggest that Lpb. pentosus SM1 has promising applications as a probiotic candidate for promoting gut health and preventing pathogenic infections.