The anticancer and antimicrobial activity associated with the thiazolidinone framework, several new (Z)-5-(substituted)-2-(methylthio) thiazol-4(5H)-one analogues 3a-l and 5a-l. All synthesized compounds were evaluated for anticancer and antimicrobial activity in vitro. Amongst these, the compounds 3c, 3f, 3h, 3j and 5a, 5c, 5d, 5e, 5f, 5h, 5j showed highest antibacterial and antifungal activity among the series. The compounds 3c and 5e exhibited significant antibacterial activity against E. coli, whereas compound 5f displayed significant antifungal activity against fungal strains i.e. A. oryzae. The in vitro anticancer studies revealed that 3i, 3k and 5b, 5k are the most active compounds against MCF-7 and BT-474 human breast cancer cell lines.