The primary treatment for hepatocellular carcinoma (HCC) involves surgical removal of the primary tumor, but this creates a favorable environment for the proliferation and spread of residual and circulating cancer cells. The development of remimazolam-based balanced anesthesia is crucial for future antitumor applications. It is important to understand the mechanisms of cytotoxicity for HCC in detail. We performed cell viability analysis, western blotting analysis, reverse transcription-polymerase chain reaction analysis, and flow cytometry analysis in two HCC cell lines, HepG2 and Hep3B cells. Our data demonstrated that remimazolam induced cytotoxicity by suppressing cell proliferation, inhibiting G1 phase progression, and affecting mitochondrial reactive oxygen species (ROS) levels, leading to apoptosis, DNA damage, cytosolic ROS elevation, lipid peroxidation, autophagy, mitochondrial depolarization, and endoplasmic reticulum stress. Inhibitors of apoptosis, autophagic cell death, and ferroptosis and a ROS scavenger failed to rescue cell death caused by remimazolam besylate. Our combination index revealed that remimazolam besylate has the potential to act as a sensitizer for targeted tyrosine kinase inhibitor therapy for HCC. Our findings open up new possibilities for combinatory HCC therapy using remimazolam, leveraging its dual functional roles in surgery and drug therapy for liver cancers.