The aim of this study is to investigate the physicochemical behavior of some selected thiobarbituartes and explore their interaction with ds-DNA. To the best of our knowledge limited work has been reported on physicochemical investigation of these medicinal compounds and their interaction with ds-DNA. Therefore, this lack of information on the fundamental physicochemical studies of such medicinal compounds needs to be addressed. For this purpose five thiobarbiturate derivatives, denoted as: MKA 25, MKA26, MKA27, MKA28, MKA29, have been synthesized and characterized. Various physicochemical investigations of these thiobarbiturates are carried out. The thermal decomposition of these medicinal compounds was also studied by TGA technique. Further the mode of interaction and binding of these newly synthesized compounds with ds-DNA was carried by using UV–Vis spectrometry. It was seen from the overall results that the physicochemical properties as well as the extent of their interaction with biopolymer not only depend on the side alkyl groups but also on varying the functional groups of the samples.