<p>This study investigated the complexation behavior between different cyclodextrins (CDs) and various preservatives used in topical ocular formulations, including benzyl alcohol, phenethyl alcohol, benzalkonium chloride, and sorbic acid. The investigations were conducted using nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), and heat capacity analysis. NMR data revealed distinct chemical shift changes, indicating strong interactions between the aromatic rings of the preservatives and the CD cavities, with β-CD demonstrating the most significant shifts for benzyl alcohol and phenethyl alcohol, and α-CD exhibiting the strongest interaction with benzalkonium chloride and sorbic acid. ITC analysis confirmed varying complexation constants, with benzalkonium chloride showing the highest affinity towards CDs, particularly α-CD, while γ-CD demonstrated the weakest binding across all preservatives. Temperature dependence studies showed that the complexation constant for benzalkonium chloride and sorbic acid with α-CD decreased with increasing temperature, a trend supported by van’t Hoff analysis, which indicated a correlation between theoretical and empirical data for these systems. Heat capacity measurements revealed predominantly negative values, indicative of hydrophobic interactions driving complexation, except for benzyl alcohol complexed with α-CD, which showed a positive heat capacity due to conformational changes. Overall, this study highlighted the differential binding affinities of preservatives with CDs, with β-CD forming the most stable complexes with benzyl alcohol and phenethyl alcohol, and α-CD showing the strongest interactions with benzalkonium chloride and sorbic acid, emphasizing the impact of structural features and temperature on inclusion complex formation and thereby a physical pharmaceutical input when formulating topic ocular formulations that would need solubilization applying CDs and preservation.</p>