Human adenovirus (HAdV) is a significant viral pathogen that causes severe acute respiratory infections (SARIs) in children and immunocompromised patients. Currently, no specific treatment options are available for HAdV infections. This study used a green fluorescence protein-based, high-throughput screening (HTS) assay on a botanical drug library containing 3,697 botanical compounds to identify agents that could inhibit HAdV. Four compounds with anti-HAdV-C5 activity in the low-micromolar range were identified and inhibited other wild-type HAdVs known to cause SARIs. Among these compounds, 13-methylberberine chloride presented the highest select index values. Berberine is a commercially available natural product-derived isoquinoline alkaloid with multiple pharmacological effects and is widely used in Asian countries. We systematically evaluated the anti-HAdV activity of six berberine-derived compounds in vitro and performed a time-of-drug-addition assay to explore their antiviral modes of action. Mechanistic studies revealed that berberine and its analogs inhibit HAdV replication by downregulating the MAPK signaling pathway, particularly ERK activation, which is crucial for viral replication and progeny production. Our findings suggest that berberine is a promising candidate for the development of anti-HAdV therapies.