To elucidate whether milk oligosaccharides can improve antibiotic-associated diarrhea, the effects of milk oligosaccharides on diarrhea, intestinal flora, and metabolic functioning were investigated in an antibiotic-associated diarrhea C57BL/6J mouse model undergoing treatment with ampicillin. Mice were divided into a normal control group, a model group, and low-and high-dose milk oligosaccharide groups, and one week after intervention, the diarrhea phenotype, inflammatory factor levels, tight junction protein expression levels, and intestinal flora and short-chain fatty acid metabolism of the mice were analyzed through 16S rRNA high-throughput sequencing and gas chromatography. The results showed that, compared to the model group, the body weight and AQP3 levels of the high-dose milk oligosaccharide group increased by 8.65%, and 128.46%, respectively, along with a decrease of 24.51% in the fecal moisture content and respective reductions of 26.98%, 30.31%, and 37.60% in the IL-6, TNF-α, and IL-1β levels and an increase of 59.21% in the IL-10 level. The mRNA expression levels of the tight junction proteins ZO-1, claudin-1 and occludin were significantly higher in the milk oligosaccharide group than the MC group, whereas Akkermansia, Bacteroidetes, Parabacteroides, and Duberella were significantly enriched in the high-dose milk oligosaccharide group. The acetic acid, propionic acid, and butyric acid contents in the intestines of mice in the high dose bovine milk oligosaccharide group were 2.18, 2.31, and 2.25 times that of those obtained for the MC group, respectively. We conclude that the milk oligosaccharides negated antibiotic-associated diarrhea by improving the diarrhea phenotype, regulating the intestinal inflammatory response, promoting the intestinal barrier function, enriching beneficial bacteria, and producing short-chain fatty acids in the mice. This study provides theoretical support for the application of milk-derived functional ingredients in the development of food or health products for those suffering from diarrhea.