Background <p>Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and one of the leading causes of cancer-related deaths worldwide. Early diagnosis of HCC, as well as the identification of biomarkers that can help determine the effectiveness of applied treatments, is extremely important. In the present study, the effects of Theranekron (TCAE, Tarantula cubensis alcoholic extract) and Sorafenib (S) on plasma and liver tissue levels of alpha-fetoprotein (AFP), heat shock protein 90 alpha (HSP90α), and indoleamine 2,3-dioxygenase 1 (IDO1) were investigated in Wistar-Albino rats with experimentally induced HCC using diethylnitrosamine (DEN) and n-nitrosomorpholine (nMOR).</p> Methods <p>The study included a total of 58 rats divided into 7 groups: Healthy Group (HG, <i>n</i> = 6), HG + TCAE (HGT, <i>n</i> = 6), HG + Sorafenib (HGS, <i>n</i> = 6), HCC Control (HC, <i>n</i> = 10), HCC + TCAE (HT, <i>n</i> = 10), HCC + Sorafenib (HS, <i>n</i> = 10), and HCC + TCAE + Sorafenib (HTS, <i>n</i> = 10).</p> Results <p>Plasma and liver AFP, HSP90α, and IDO1 levels were found to be significantly elevated in the HC group compared with the healthy control groups (<i>p</i> &lt; 0.001). Administration of Theranekron and Sorafenib, either alone or in combination, resulted in significant reductions in AFP, HSP90α, and IDO1 levels, particularly in plasma (<i>p</i> &lt; 0.001). Furthermore, histopathological evaluation revealed that Theranekron and Sorafenib treatments exhibited less tumorigenic morphology compared with the HC group.</p> Conclusion <p>Taken together, these results demonstrate that Theranekron enhances the antitumor efficacy of Sorafenib. Moreover, HSP90α and IDO1 levels appear to provide higher specificity and sensitivity, suggesting their potential as more reliable biomarkers for monitoring tumor prognosis.</p>