A series of 1,3,5-trisubstituted pyrazolines based homoleptic Ru(III) complexes of type [Ru(L^1–7)₃]·(PF₆)₃ (L^1–7 = pyrazoline ligands) have been synthesized and characterized by elemental analysis, electronic spectroscopy, conductance measurements, thermogravimetric analysis (TGA), electron paramagnetic resonance (EPR), fourier transform infrared (FT-IR) spectroscopy and liquid chromatography mass spectroscopy (LC-MS). Octahedral geometry around ruthenium has been assigned in all complexes using EPR and electronic spectral analysis. All complexes have been investigated for their interaction with Herring Sperm (HS) DNA utilizing an absorption titration (K_b = 2.42–6.07 × 10⁵ M⁻¹) and viscosity measurement study. The studies suggest the classical intercalative mode of binding. The DNA-binding property of the Ru(III) complexes was also investigated theoretically using a molecular docking study and suggests an intercalation binding mode between the complex and nucleotide base pairs. A cleavage study on pUC19 DNA has been performed by agarose gel electrophoresis. The results indicated that the Ru(III) complexes can more effectively promote the cleavage of plasmid DNA. The free ligands and their complexes have been evaluated for cytotoxicity activity against S. pombe cells at a cellular level. A comparative study of cellular level cytotoxicity values of the all compounds indicates that the metal complexes show better activity against S. pombe cells compared to the pyrazoline ligands. The complexes have been screened for their in vitro antibacterial activity against two Gram(+ve) and three Gram(−ve) microorganisms. Ru(III) complexes are good in vitro cytotoxic agents and 50% lethal concentration (LC₅₀) values are in range of 5.296–7.925 μg mL⁻¹. All newly synthesized Ru(III) complexes have been also evaluated for their in vitro antimalarial activity against Plasmodium falciparum strain [inhibition concentration (IC₅₀) = 0.54–0.92 μg mL⁻¹].