Liver injury is a globally serious problem that may be observed post-chemotherapeutic administration in chronic crises such as tuberculosis (TB). Rifamycin (RIF), particularly, is an indispensable treatment regimen for TB with a significant negative hepatic impact. Therefore, this research aims to assess the restorative role of melatonin (MEL) against rifampicin (RIF)-associated hepatic damage in rats. Moreover, to investigate the ultimate mechanism of the antioxidant potential of MEL with multimodal assessment and in silico molecular conformation. Adult male Wistar albino rats were weighed and divided into four groups of ten rats each. The control group received the vehicle (0.5 ml/day), the RIF-intoxicated group (100 mg/kg/day orally), the MEL-treated group (10 mg/kg/day intraperitoneally), and the Co-administered RIF and MEL group at the same regimen for 21 consecutive days. Blood and hepatic tissue samples were obtained for biochemical, histological, and molecular studies. High in vitro antioxidant scavenging potential of MEL with an IC of 94.66 µg/ml was attained using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS) analyses of MEL indicated the presence of mainly cinnamic acid, m-(trimethyl-silyl ester), in addition to several reactive antioxidant moieties. The plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lipid profiles were significantly reduced in the MEL + RIF co-administered group in contrast to the RIF-treated group (P < 0.05). Moreover, MEL significantly decreased the hepatic oxidative biomarkers malondialdehyde (MDA) and significantly increased the estimated antioxidant enzyme levels (P < 0.05) of superoxide dismutase (SOD) and glutathione peroxidase (GPX). Histological evaluations revealed mild hepatic injuries and inflammatory cellular infiltration in MEL-treated animals. Molecular docking explored the high-affinity interaction of MEL and cinnamic acids with hepatic cytochrome P450 isoform 3A4 (CYP3A4) protein target, confirming their hepatic restorative potential. This study endorses the antioxidant potential role of MEL in RIF-hepatic injury.