Homoisoflavonoids (3-benzylidenechroman-4-ones) are related to flavonoids and occur as natural products and exhibit various biological activities. Because of these properties, organic chemists are very interesting to homoisoflavonoids. The present study was designed to examine the interactions of four synthetic homoisoflavonoids (BMC, BPC, HBC and HBMC) that exhibit antioxidant, antifungal and antiviral activities, with calf-thymus DNA and RNA in aqueous solution at physiological conditions, using constant DNA and RNA concentration and various homoisoflavonoids/polynucleotide (phosphate) ratios. Fourier transform infrared and UV–Visible spectroscopic methods were used to determine the homoisoflavonoids binding site, the binding constants and the stability of homoisoflavonoids–DNA and RNA complexes in aqueous solution. The affinity of homoisoflavonoids–DNA and RNA bindings are in the order of BPC>BMC>HBMC>HBC. Our spectroscopic results provide a major structural analysis of homoisoflavonoids–biopolymers interaction, which helps elucidate the nature of this biologically important complexation in vitro.