The chemical investigation of lichen Ramalina leiodea (Nyl.) Nyl. yielded five known metabolites, i.e., usnic acid (1), ethyl everninate (2), scrobiculin (3), methyl 2,6-dihydroxy-4-methylbenzoate (4) and 4-[(2-hydroxy-4-methoxy-6-propylbenzoyl)oxy]-2-methoxy-6-propylbenzoic acid (5). To develop compound libraries on 4, a series of semi-synthetic derivatives was prepared (4a––e). All the metabolites and semi-synthetic analogues were screened for antimicrobial and anti-tubercular activities. The results showed that compounds 3 and 5 were very active against antibacterial and antifungal strains, while the semi-synthetic analogues 4a–e are moderately active on all tested microbial strains. In addition, compounds 4b and 4d showed better antimycobacterial activity with MIC value of 1.6 µg mL-1, than streptomycin with an MIC of 6.25 µg mL-1 against M. tuberculosis. All the semi-synthetic analogues exhibited better anti-tubercular activity than the isolated metabolites. This is the first report on the synthesis and biological activities of these novel benzohydrazide derivatives. benzohydrazides, derivatization, Mycobacterium tuberculosis, well plate method