The modulation of the prototropic equilibrium of a cancer cell photosensitizer, norharmane (NHM), within a niosome microheterogeneous environment has been investigated. The contrasting effects of temperature and extrinsically added salt on the photophysics of niosome-bound drug have been meticulously explored from steady-state and time-resolved spectroscopic techniques. The cation reversible arrow neutral prototropic equilibrium of NHM is found to be preferentially favored toward the neutral species with increasing salt concentration, and the results are rationalized on the basis of water penetration to the hydration layer of niosome. The effects are typically reversed with temperature. The differential rotational relaxation behavior of NHM under various conditions has also been addressed from fluorescence anisotropy decay. Further, the study delineates the application of beta-cyclodextrin (beta CD) as a potential host system, leading to drug sequestration from the niosome-encapsulated state. To this end, a detailed investigation of the thermodynamics of the niosome:beta CD interaction has been undertaken by isothermal titration calorimetry (ITC) to unravel the notable dependence of the thermodynamic parameters on temperature. Consequently, a critical analysis of the variation of the enthalpy change (Delta H) of the process with temperature leads to the unique observation of a positive heat capacity change (Delta C-p) marking the hallmark of hydrophobic hydration.