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• A new series of benzohydrazide derivatives having 2-bromo-1-(4-fluorophenyl)ethanone were synthesized. • The synthesized compounds were screened for antimicrobial activity and evaluated for in silico screening against Thymidylate kinase (PDB ID: 1G3U) and Thioesterase of Polyketide Synthase (PDB ID: 5V3Y). • All the synthesized compounds measured for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv and 4 h displayed more than 99% growth inhibition. • From 4a-j, 4 h exerted strong interaction with 5V3Y, it is concluded that 4 h might exert its vital role as an antitubercular agent through inhibition of thioesterase activity of polyketide synthase Pks13 enzyme. Tuberculosis scourge is at the most noteworthy danger, and it is urgent to characterize new defenses against it. A new series of benzohydrazide derivatives containing benzylidene benzohydrazide linkage with 2-bromo-1-(4-fluorophenyl)ethanone via methylene-oxy group. The characterization of benzohydrazide derivatives was performed by 1H and 13C NMR, FT-IR and mass spectrometry. The synthesized compounds were screened for antimicrobial activity and evaluated for in silico screening. All the synthesized compounds measured for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv and 4 h displayed more than 99% growth inhibition. [Display omitted] [ABSTRACT FROM AUTHOR]