Four series of heterocyclic compounds, namely, tetrahydro-2H-1,3,5-thiadiazine thione derivatives were synthesized in good to excellent yields and were screened for their in vitro antileishmanial activities against Leishmania major (promastigotes). Most of the compounds showed significant antileishmanial activity within the range of IC50 = 15.48-39.36 μM when compared with standard pentamidine (IC50 = 14.95 μM). The structure-activity relationship showed that N-3 and N-5 substituents have a key role against leishmanicidal activity. The ester analogues (series B) were found to have a 1.5 to 5-fold reduced activity compared to their acidic counterparts. Cytotoxicity against mammalian mouse fibroblast 3 T3 cells was also evaluated and compared between the acid and its ester analogue. The reduction of antileishmanial activity and loss of toxicity in the newly developed THTT ester derivative indicates that these compounds can be used as a template study for the production of effective antileishmanial ester prodrugs.