The application of probiotics, in aquaculture, is becoming increasingly widespread and have had positive application effects. However, reports of loach-derived probiotics are quite limited. In this study, two representative strains of lactic acid bacteria with excellent traits, namely, Weissella confusa N17 and Lactobacillus saniviri N19, were screened from the intestine of healthy loaches. W. confusa N17 and L. saniviri N19 could inhibit different common various pathogenic bacteria, especially Aeromonas spp., and were sensitive to the most common antibiotics. The survival rate of the two strains exceeded 50% after 4 h of incubation in 10% loach bile. Moreover, the two strains showed significant tolerance to trypsin. Their autoaggregation capacity and hydrophobicity were greater than 30%. In addition, the aggregation ability of both strains was higher than 30% for both A. veronii TH0426 and A. hydrophila TPS. The two strains had a high biofilm-forming ability and strong adhesion to epithelioma papulosum cyprini (EPC) cells. Scanning electron microscopy results showed that the culture supernatants of the two strains had a significantly destructive effect on A. veronii TH0426 and A. hydrophila TPS. Overall, the traits of W. confusa N17 were better than those of L. saniviri N19. Genome sequencing and analysis demonstrated a lack of virulence factor-related or drug resistance-related genes in genome N17. The diet supplemented with the W. confusa N17 strain significantly improved the resistance of loaches to A. veronii infection, and the protection rate reached 57.1%. Therefore, W. confusa N17 exhibits promising for further applications in loach aquaculture.