Ultrasonic technique is employed for the investigation of molecular interactions in aqueous solutions of antibiotic norfloxacin for different molar concentrations around the physiological temperatures. The measured and computed thermo acoustic parameters at atmospheric pressure, confirm the existence of cluster formation and hydrogen bonded interaction of norfloxacin at 303K while existence of dispersed state of norfloxacin and the dominance of solute-solvent interaction at 310K and 313K. For further confirmation, the stacking parameter (Δ) was obtained through the Hemme's isodesmic model. The values of ‘Δ’ claim the dimeric form with stacking behaviour of norfloxacin at ambient temperature and dispersed monomeric state of norfloxacin in the aqueous environment which enable for its drug action around physiological temperature. The velocity derivative limiting slope (A) and stacking constant (K) values support these observations. The structure change of solvent is discussed based on the deviation of adiabatic compressibility of solutions from the solvent. A detailed quantum chemical calculations using Gaussian 09 software program were performed. It is found that in addition to the hydrogen bonding interactions, stacking interaction play a vital role in deciding the associative behaviour of norfloxacin molecules.