In the present work a set of five Ni(II) complexes with general formula [Ni(L)2] and with HL = N-thenoylthiocarbamic-O-n-alkylylesters (n = 2–6) has been prepared and characterized in solution by UV–vis and NMR spectroscopies. Three of them were also characterized in the solid state by X-ray diffractometry. The energy rotation of the thiophene ring of ligand was evaluated theoretically. Liposomes of complexes were prepared in order to evaluate their ability to interact with the membrane. Furthermore, their biological activities were evaluated in a set of bacteria (gram+ and gram−) and yeasts. The X-ray structure determination confirms that bidentate ligand forms a tetra co-ordinated complex with an S2O2 co-ordination sphere around the nickel(II) ion in a cis configuration. The metal centre is coordinated in a square planar fashion. NMR spectra taken in solution show a diamagnetic signal compatible with a square-planar geometry around the metal centre. The values obtained for the liposome/water partition coefficients (Kp) show that [Ni(ttete)2] and [Ni(ttpre)2] have a similar membrane partition ability, whilst the [Ni(ttbue)2] derivative presents a significantly higher Kp, describing a stronger interaction within the membrane. For all the compounds, [Ni(ttpre)2] has a higher efficacy against Gram negative bacteria and yeasts nevertheless, the anti-yeast and anti-bacterial activity values of all tested compounds are lower than ones of the reference compounds.