Macromolecular crowding, manifested by high concentrations of proteins and nucleic acids in living cells, significantly influences biological processes such as enzymatic reactions. Studying these reactions in vitro, using agents such as polyetthylene glycols (PEGs) and polyvinyl alcohols (PVAs) to mimic intracellular crowding conditions, is essential due to the notable differences from enzyme behaviors observed in diluted aqueous solutions. In this article, we studied Mycobacterium tuberculosis (Mtb) DNA gyrase under macromolecular crowding conditions by incorporating PEGs and PVAs into the DNA supercoiling reactions. We discovered that high concentrations of potassium glutamate, glycine betaine, PEGs, and PVA substantially stimulated the DNA supercoiling activity of Mtb DNA gyrase. Steady-state kinetic studies showed that glycine betaine and PEG400 significantly reduced the KM of Mtb DNA gyrase and simultaneously increased the Vmax or kcat of Mtb DNA gyrase for ATP and the plasmid DNA molecule. Molecular dynamics simulation studies demonstrated that PEG molecules kept the ATP lid of DNA gyrase subunit B in a closed or semiclosed conformation, which prevented ATP molecules from leaving the ATP-binding pocket of DNA gyrase subunit B. The stimulation of the DNA supercoiling activity of Mtb DNA gyrase by these molecular crowding agents likely results from a decrease in water activity and an increase in excluded volume.