Background: A bioassay-guided fractionation technique was used to evaluate the active constituents of the perennial plant L. officinale W.D.J. Koch (Apiaceae) against multidrug resistant (MDR) Mycobacterium tuberculosis. Methods: Column chromatography was used to isolation of compounds from L. officinale and spectroscopic methods including 1D and 2D NMR (Nuclear magnetic resonance) and HRMS (high resolution mass spectrometry) were used to identification of the isolated compounds. Also, to evaluate antibacterial activity, minimum inhibitory concentration (MIC) was carried out by broth micro-dilution method. Finally, molecular docking (MD) was performed using the Schrödinger package to evaluate interactions between the active compounds and InhA protein. Results: Phytochemical analysis of the ethyl acetate extract of the plant roots led to isolation of bergapten (1), isogosferol (2), oxypeucedanin (3), oxypeucedanin hydrate (4), imperatorin (5), ferulic acid (6) and falcarindiol (7). Falcarindiol and oxypeucedanin indicated a moderate activity on MDR M. tuberculosis with MIC values of = 32 and 64 μg/mL, respectively. Antibacterial activity of falcarindiol was also observed against S. aureus and methicillin-resistant S. aureus strains with the MIC values of 7.8 and 15.6 μg/mL, respectively. The results of docking analysis showed a good affinity of oxypeucedanin (3) and falcarindiol (7) to InhA enzyme with docking score values of -7.764 and -7.703 kcal/mol, respectively. Conclusion: Finally, 7 compounds were isolated from L. officinale that compounds 2-6 report for the first time from this plant. On the basis of the molecular docking (MD) study, oxypeucedanin (3) and falcarindiol (7) as active compounds against M. tuberculosis may be proposed as potential inhibitors of 2-trans-enoyl-ACP reductase (InhA), a key enzyme involved in the biosynthesis of the mycobacterial cell wall. Moreover, antibacterial activity of falcarindiol against methicillin-resistant S. aureus (MRSA) was remarkable.