Tuberculosis is an ancient disease of mankind, and its causative bacterium is Mycobacterium tuberculosis. Isoniazid is one of the most effective first-line anti-tuberculosis drugs. As prodrugs, it and its derivative ethionamide act on enoyl-acyl carrier protein reductase (InhA) after being oxidized in bacteria, and kill the bacteria by inhibiting the formation of Mycobacterium tuberculosis cell walls. However, the S94A mutation of InhA causes Mycobacterium tuberculosis to develop cross-resistance to isoniazid and ethionamide. This work is dedicated to studying the cross-resistance mechanism of isoniazid and ethionamide through theoretical calculations. First, thermodynamic integral simulations are used to accurately calculate the relative binding energy of two drugs in the mutant and wild type system. Furthermore, through classic molecular dynamic simulations and MM-GBSA calculation, some key residues are identified and the binding affinity of isoniazid and ethionamide reduced by 9–13 kcal/mol due to S94A mutation. The hydrogen bond between Ala94 and isoniazid (ethionamide) disappeared and the energy contribution of Ala94 decreased after the mutation. In addition, the dynamic network analysis indicated that the mutation of Ser94 also indirectly affected the conformation of key residues such as Met147, Thr196 and Leu97, resulting in a reduction in the energy contribution of these residues. Finally, the binding conformation of isoniazid and ethionamide has also undergone major changes. The obtained results could provide valuable information for the future molecular design to overcome the drug resistance.