This study evaluated the effects of antimicrobial peptide (AMP) and tributyrin (TB) on dairy calves in terms of growth performance, immunity, oxidative stress, and intestinal microflora. A total of 40 female calves were divided into four treatment groups ( = 10): basal diet +0.015% essential oil, basal diet +0.03% AMP, basal diet +0.15% TB, and basal diet +0.03% AMP + 0.15% TB. AMP and TB supplementation increased the average daily gain (ADG) and weaning weight, while reducing diarrhea occurrence. Additionally, AMP and TB supplementation reduced the levels of reactive oxygen species (ROS) and malonaldehyde (MDA), while increasing superoxide dismutase (SOD) levels and serum immunoglobulin M (IgM) levels. However, the combined use of AMP and TB did not significantly affect the average daily feed intake, ADG, weaning weight, or diarrhea incidence but decreased ROS levels, while increasing SOD levels as well as MDA and IgM levels. Moreover, AMP and TG supplementation increased the relative abundance of several beneficial fiber- and mucin-degrading bacteria in the gut, in contrast to combined AMP and TB supplementation. The 16S rRNA results showed that AMP supplementation significantly increased the relative abundance of , and ( < .01), and significantly decreased the relative abundance of and ( < .01). The TB supplementation significantly increased the abundances of and ( < .01), and significantly decreased the relative abundances of , and ( < .01). The combined use of AMP and TB significantly increased the relative abundance of and ( < .01), and significantly decreased the relative abundance of , and ( < .01). In summary, diets supplemented with either AMP or TB improved the intestinal microflora, growth performance, and health of weaned calves, but combined use was detrimental to calf performance.