Fatty acids of specific chain lengths have been shown to inhibit the growth of Mycobacterium tuberculosis. In the present study, specific synthetic aromatic derivatives of n-octyl esters were investigated for their property to inhibit the growth of M. tuberculosis H37Ra. Agar well diffusion assay indicated that the crude synthetic derivatives obtained by the esterification of phthalic acid (PA) and n-octanol exhibited antimycobacterial activity. Further, the activity was authenticated with the Miroplate Alamar Blue Assay (MABA). Subsequently, the active component was purified by bioactivity guided chromatographic fractionation. The structure of the synthetic derivative was deduced by UV–Vis, FT-IR, LC–MS, GC–mass spectrometry, and NMR spectroscopy. Molecular docking and molecular dynamic simulation (MDS) were performed with Autodock 4.0 and GROMACS 5.1.2 softwares, respectively. It was found that mono-n-octyl phthalate (MOP) exhibited antimycobacterial activity with a MIC of 20 μg/mL, and not by any other related compounds, including di-n-octyl phthalate, PA, phthalic anhydride, and n-octanol. Binding of MOP with protein kinase B can participate in the binding cavity region, which was previously reported. Subsequently, we authenticate the stability with MDS. This is first report on the inhibition of M. tuberculosis growth by MOP. © 2018 International Union of Biochemistry and Molecular Biology, Inc.