The present work demonstrates the photophysical characterization of the interaction of a promising cancer cell photosensitizer, harmane (HM), with biomimetic micellar nanocavities having varying surface charge characteristics. The polarity-sensitive prototropic transformation of HM is remarkably modified upon interaction with the macromolecular assemblies of micellar systems and is manifested through significant modulations on the absorption and emission profiles of HM. The ground- and excited-states prototropic equilibria of HM are found to be differentially modulated in various micellar assemblies. Out of various possibilities to assess the drug (HM)-micelle interaction mechanism, the postulate of varying extent of drug penetration into micellar units depending on the compactness of their headgroup arrangements is found to suitably rationalize and correlate different experimental findings, including the differences in binding constant (K) and free energy change (δG) of the interaction process. The micropolarity measurement has been exploited to evaluate the probable binding location of the drug which reveals that the cationic drug molecule does not penetrate deep into the micellar core region and the results are further substantiated from fluorescence quenching experiments. The work also pays proper attention to delineate the modulation in dynamical behaviors of the drug following interaction with the micellar systems. Wavelength-sensitive fluorescence parameters reveal the slower rate of solvent-relaxation around the excited probe within the micelle-encapsulated microheterogeneous environments. The enhancement of fluorescence anisotropy and rotational relaxation time of the drug in micellar environments from that in pure aqueous buffer suggests entrapment of the drug in motionally constrained regions introduced by the micelles. © 2012 American Chemical Society.