Cognitive dysfunction (CD) associated with cholinergic deficits is a hallmark of Alzheimer’s disease (AD) and related disorders. The natural edible herb Anoectochilus roxburghii has various pharmacological activities, including anti-inflammatory, antioxidant, and neuroprotective effects. However, the underlying mechanism by which A. roxburghii alleviates CD via the microbiota–gut–brain (MGB) axis remains unclear. In this study, a scopolamine-induced CD mouse model was used to evaluate the therapeutic effect of A. roxburghii aqueous extract (ARAE) through behavioral tests, intestinal and brain histopathology, ELISA quantitative assays, gut microbiota sequencing, and metabolomics analysis. ARAE effectively improved cognitive and memory abilities in the mouse model, as demonstrated by significantly reduced escape latency and increased crossings over the target platform in the Morris water maze test. Mechanistic studies elucidated that ARAE effectively reduced the release of inflammatory factors, alleviated oxidative stress, and corrected cholinergic imbalance. Furthermore, ARAE reshaped the gut microbiota and upregulated the expression of tight junction proteins claudin-1, occludin, and zonula occludens-1 (ZO-1), as well as mucin-2 (MUC-2), thereby contributing to the integrity of the intestinal barrier. Meanwhile, ARAE increased the levels of propionic acid and butyric acid, concurrently restoring the disordered host–microbiota co-metabolism by targeting the tryptophan metabolism pathway. In summary, ARAE alleviates scopolamine-induced CD via the MGB axis through anti-inflammatory, antioxidant, cholinergic, and gut barrier-enhancing mechanisms. These findings indicate that A. roxburghii may be a promising candidate for the treatment of CD associated with cholinergic dysfunction and lay a foundation for further research in more comprehensive AD models.