Over the past two decades, gut dysbiosis has gained significant attention due to its multifaceted involvement in diverse health disorders. Gut barrier dysfunction increases intestinal permeability, which allows gut-derived toxicants into the systemic circulation and contributes to adverse health outcomes. The proteins, including zona occludens 1 (ZO-1), occludin, and claudin 4, maintain the integrity through forming a tight junction complex and restricting the entrance of noxious substances through the intestinal epithelial apical layer. It had been reported that the reduction in the relative expression of mucosal junctional proteins can trigger a broad spectrum of disorders, including inflammatory bowel diseases, renal impairment, neurocognitive dysfunctions, autoimmune disorders, colorectal cancer, and others. Preclinical studies demonstrate the role of puerarin in restoring the intestinal barrier function through increasing the levels of tight junction proteins and exerting anti-inflammatory and antioxidant effects. Furthermore, previous scientific evidence suggested the deteriorating effects of higher concentrations of adenine on the intestinal barrier functions. This study aims to elucidate the molecular docking of puerarin and adenine against the ZO-1, occludin, and claudin 4. Puerarin shows a lesser binding energy against ZO-1 (−7.187 kcal/mol) and claudin 4 (−9.193 kcal/mol), whereas no significant results were obtained in the case of occludin. They exhibit stronger hydrophobic interaction and hydrogen bonding to the amino acid residues present at the active site of the ZO-1 and claudin 4. In contrast, adenine displayed fewer interactions, likely due to its inhibitory effects at the active site pockets of ZO-1, occludin, and claudin 4. These findings suggest that puerarin may have therapeutic potential in modulating tight junctional proteins. However, further in vitro and in vivo studies are required to determine the preventive effects against gut dysbiosis-associated health complications and intestinal disorders.