New steroidal imidazolidinthione derivatives (4-6) were synthesized from steroidal thiosemicarbazones and dichloroethane. The synthesized compounds were characterized using spectral data analysis. Theoretical DFT involving B3LYP/6-31G** level of theory was employed to gain insights into the molecular structure of the target compounds. MEPS and FMO analysis were carried out. HOMO-LUMO energy gap was determined which helped to evaluate various global descriptors like hardness, chemical potential, electronegativity, nucleophilicity and electrophilicity index, etc. The calculated properties established that the synthesized products are more or less similar in their reactivity behaviour. To explore their biological potential, interaction studies of compounds (4-6) with DNA were carried out using various biophysical techniques. The compounds bind DNA preferentially through electrostatic and hydrophobic interactions with K-b of 3.21 x 10(3) M-1, 2.79 x 10(3) M-1 and 2.26 x 10(3) M-1, respectively indicating the higher binding affinity of compound 4 towards DNA. Gel electrophoresis of compound 4 demonstrated strong interaction during the concentration dependent cleavage activity with pBR322 DNA. It was observed that these steroidal imidazolidinthiones are minor groove binders of DNA which was validated using molecular docking studies. An in vitro cytotoxicity screening using MTT assay revealed that the compounds (4-6) exhibit potential toxicity against different human cancer cells. Highest antiproliferative effect was observed on HeLa cells by compound 4. The results suggested that compounds 4-6 cause apoptotic cell death by cleaving apoptotic protein caspase-3 and suppress anti-apoptotic protein Bcl-2 in HeLa cancer cells.