A series of square planar cyclometalated heteroleptic platinum(II) complexes of the type [(C^N)Pt(O^O)] [where, O^O is a β‐diketonato ligand of acetylacetone (acac), C^N = cyclometalating 7‐(4‐fluorophenyl)‐5‐phenylpyrazolo[1,5‐a]pyrimidine (L1), 7‐(4‐chlorophenyl)‐5‐phenylpyrazolo[1,5‐a]pyrimidine (L2), 7‐(4‐bromophenyl)‐5‐phenylpyrazolo[1,5‐a]pyrimidine (L3), 7‐(4‐methoxyphenyl)‐5‐phenylpyrazolo[1,5‐a]pyrimidine (L4), 5‐phenyl‐7‐(p‐tolyl)pyrazolo[1,5‐a]pyrimidine (L5)] have been design, synthesized and characterized. All compounds have been screened for biological studies like in vitro antibacterial, in vitro cytotoxicity, cellular level cytotoxicity, absorption titration, viscosity measurements, fluorescence quenching analysis, molecular docking and DNA nuclease. The intrinsic binding constants (Kb) of compounds with HS‐DNA has been obtained in range of 2.892–0.242 × 105 M−1. All the compounds bound with HS DNA by partial intercalative mode of binding. MIC study has been carried out against Gram(+ve) and Gram(−ve) bacterial species. In vitro cytotoxicity against brine shrimp lethality bioassay has been also carried out. The LC50 values of the ligands and complexes have been found in range of 56.49–120.22 μg/mL and 6.71–11.96 μg/mL, respectively.