The velocity of ultrasonic waves (U), density (ρ) and viscosity (η) of o-nitrophenol (o-NP) with methyl acetate (MA) in benzene was measured at 303 K in seven different weight fractions. The other related acoustic and thermodynamic parameters like adiabatic compressibility (β), free length (Lf), free volume (Vf), internal pressure (πi), and Gibb’s free energy (ΔG) were acquired. The obtained parameters are played a significant role in studying the basis of the molecular interactions. The FTIR analysis was carried out to identify the functional groups of the mixture o-NP + MA. The optimized geometry of the mixture o-NP + MA is analyzed extensively by the DFT method. In addition, non-covalent interactions were confirmed by the Multiwave function and VMD software packages. The possibilities of H-bonding formation between o-nitrophenol and methyl acetate molecules were identified and their corresponding hydrogen bond wavelengths were calculated using Gaussian software. The FTIR spectra of pure o-NP, pure MA and a mixture of o-NP + MA were recorded, which confirms the existence of non-covalent interactions (NCI’s) within the solute species. The frequency deviations in the functional groups of the mixture are analyzed with characteristic regions. The identified NCI’s are validated by ultrasonic and DFT analysis.