This study investigated the effects of mulberry (Morus alba L.) leaf flavonoids and Candida tropicalis on the growth, gastrointestinal development, enzyme activity of gastrointestinal digesta, and blood antioxidant function of dairy calves challenged with Escherichia coli O141:K99. A total of 15 Holstein preweaning bull calves (age = 28 d; body weight = 51 ± 3.9 kg, mean ± standard deviation) were randomly divided into 3 homogeneous groups with no supplementation (control, CON), Candida tropicalis added at 1 g/calf per day (CT), and mulberry leaf flavonoids added at 3 g/calf per day (MLF). The feed efficiency was greater in calves fed MLF than in CON either before or after the E. coli challenge test. The fecal score increased after E. coli challenge, but it was greater in the CON group than in the CT treatment and in the MLF group at d 1, 2, and 3 after the E. coli challenge. Calves fed the CT diet showed decreased blood β-hydroxybutyrate concentrations compared with those on the CON and MLF diets before the E. coli challenge. Dietary supplementation with CT resulted in lower blood superoxide dismutase activity than the CON after E. coli challenge. The calves fed CT and MLF showed much lower blood glutathione peroxidase activity than the CON group after being challenged with E. coli. The tunica mucosa of the duodenum was lower in CT and MLF than in CON. The lactase activity of the jejunal digesta was greater in MLF than in CON and CT. In summary, MLF and CT supplementation might effectively improve antioxidant function and reduce the incidence of oxidative stress after challenge with E. coli in 28-d-old preweaning calves, and this effect may be partially explained by the relative decreases in blood superoxide dismutase and glutathione peroxidase as well as fecal score.