The preparation of palladium(II) complexes of 3,5-diacyl-1,2,4-triazole bis(thiosemicarbazone) (H 2 L 2 ), 2,6-diacylpyridine bis(thiosemicarbazone) (H 2 L 3 ) and benzyl bis(thiosemicarbazone) (H 2 L 4 ) is described. The new complexes [PdCl 2 (H 2 L 2 )] (1), [PdCl 2 (H 2 L 3 )] (2) and [PdL 4 ]·DMF (3) have been characterized by elemental analyses and spectroscopic studies (IR, 1 H NMR and UV–Vis). The crystal and molecular structure of PdL 4 ·DMF (L=bideprotonated form of benzyl bis(thiosemicarbazone)) has been determined by single-crystal X-ray diffraction: green triclinic crystal, a=10.258(5), b=10.595(5), c=11.189(5) Å, α=97.820(5), β=108.140(5), γ=105.283(5)°, space group P1, Z=1. The palladium atom is tetracoordinated by four donor atoms (SNNS) from L 4 to form a planar tricyclic ligating system. The testing of the cytotoxic activity of compound 3 against several human, monkey and murine cell lines sensitive (HeLa, Vero and Pam 212) and resistant to cis-DDP (Pam-ras) suggests that compound 3 might be endowed with important antitumor properties since it shows IC 50 values in a μM range similar to those of cis-DDP [cis-diamminedichloroplatinum (II)]. Moreover, compound 3 displays notable cytotoxic activity in Pam-ras cells resistant to cis-DDP (IC 50 values of 78 μM versus 156 μM, respectively). On the other hand, the analysis of the interaction of this novel Pd-thiosemicarbazone compound with DNA secondary structure by means of circular dichroism spectroscopy indicates that it induces on the double helix conformational changes different from those induced by cis-DDP.