A series of 2,5-disubstituted-1,4-benzoquinone derivatives were synthesized and evaluated for their antimicrobial activity. Coupling of 2,5-dihydroxybenzoic acid (gentisic acid) with the selected thiazolidine derivative using EDC and HOBt afforded the corresponding (4R)-3-(2,5dihydroxybenzoyl)-5,5-dimethyl-N-substituted benzylthiazolidine-4-carboxamide 2a-d. These compounds were subsequently oxidized with ferric chloride to afford the corresponding 1,4benzoquinones 3a-d. The reaction between 1,4-benzoquinone derivatives and the appropriate amine resulted in the targeted 2,5-disubstituted-1,4-benzoquinone derivatives 4a-l. The structure of the newly synthesized compound were confirmed by elemental microanalyses, IR and 1H NMR spectra. The antimicrobial activity of target compounds 4a-l was performed against Escherichia coli (E. coli) ATCC 25922 as Gram-negative bacteria, Staphylococcus aureus (S. aureus) ATCC 19433 as Gram-positive bacteria and Candida albicans (C. albicans) as yeast like fungi were determined