Salmonella typhimurium (S. typhimurium) is one of the most common Salmonella serotypes in epidemiological surveys of poultry farms in recent years. It causes growth retardation, mortality, and significant economic losses. The extensive use of antibiotics has led to the emergence of multi-drug resistance (MDR) in Salmonella, which has become a significant global problem and long-term challenge. In this study, we investigated the prevalence and features of S. typhimurium strains in duck embryos and cloacal swabs from large-scale duck farms in Shandong, China, including drug resistance and virulence genes and the pathogenicity of an S. typhimurium strain by animal experiment. The results demonstrated that a total of 8 S. typhimurium strains were isolated from 13,621 samples. The drug resistance results showed that three of the eight S. typhimurium strains were MDR with the dominant resistance profile of CTX-DX-CTR-TE-AMX-AMP-CAZ. In particular, the virulence genes invA, hilA, pefA, rck, and sefA showed high positive rates. Based on the analysis of the biological characteristics of bacterial biofilm formation and mobility, a strain of S. typhimurium with the strongest biofilm formation ability, designated 22SD07, was selected for animal infection experiments with broiler ducklings. The results of animal experiments demonstrated that infection with 22SD07 reduced body weight and bursa index but increased heart and liver indexes compared to the control group. Histological examination revealed desquamation of the intestinal villous epithelium, the presence of large aggregates of lymphocytes, and a decrease in goblet cells following infection. Furthermore, the expression of IL-10 was significantly increased in the liver at 3 dpi, while TNF-α was significantly increased in the spleen at 7 dpi. The above results indicate that S. typhimurium may pose a potential threat to human health through the food chain. This helps us to understand the frequency and characteristics of S. typhimurium in duck farms and emphasizes the urgent need to strengthen and implement effective continuous monitoring to control its infection and transmission.