New triazolothiadiazine derivatives were synthesized via the ring closure reaction of 4-amino-5-substituted-2,4-dihydro-3H-1,2,4-triazol-3-thiones with phenacyl bromides. The compounds were tested in vitro against various Candida species and compared with ketoconazole. Among these compounds, the compound bearing cyclohexyl moiety and p-chlorophenyl substituent on triazolothiadiazine ring (2i) was found to be the most potent derivative against Candida albicans (ATCC 90028). It is clear that there is a positive correlation between anticandidal activity and two functional moieties, namely cycloaliphatic group and p-chlorophenyl substituent on triazolothiadiazine ring. The compounds were also investigated for their cytotoxic effects using MTT assay. Compound 2a exhibited the highest cytotoxic activity, whereas compound 2f possessed the lowest cytotoxic activity against NIH/3T3 cells.