The density, viscosity and conductivity of binary mixtures of the front line antitubercular drug isoniazid (INH), in aqueous solution and dimethylsulfoxide (DMSO) solution, were determined at various temperatures (25, 37 and 55 °C) up to 0.3 mol⋅L−1 of INH. The apparent molar volumes were calculated from the density data. In the INH + water system the apparent molar volume of INH changed smoothly, whereas in the INH + DMSO system it passes through a maximum. Also, both systems showed pronounced maxima in their viscosity and conductivity isotherms. In addition, UV–Vis, FT-IR and 1H NMR spectroscopy were performed on the solutions. On the basis of this data, the predominant molecular interactions occurring between INH and water and between INH and DMSO were found to be hydrogen bonds. Furthermore, the susceptibility profile of DMSO, INH and its combination was studied against M. tuberculosis H37Rv and the minimum inhibitory concentration (MIC) determined. The results suggest a synergistic effect of INH at sub-MIC concentrations and DMSO.