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This study investigates the influence of fluoroquinolone drug on the colloidal behavior of cationic surfactant by using density, speed of sound, conductivity, and fluorescence spectroscopy measurements at different concentrations and temperatures. To get insight into the effect of drug on the aggregation behavior of DTAB (dodecyltrimethylammonium bromide), the critical micelle concentration (CMC) has been estimated from the conductivity and speed of sound isotherms. Interestingly, in comparison with the CMC of DTAB in water, the presence of drug is found to decrease the CMC, accounting for the fact that hydrophilic dehydration and hydrophobic interactions play a decisive role for the micellization to take place. The volumetric and compressibility parameters have been derived from the density and speed of sound used to study the nature of interactions. For fluoroquinolone drug–DTAB system, ΔG m o values are negative, which indicate that the drug-mediated ionic micelle formation process is thermodynamically feasible and spontaneous. The modulation in aggregation is further characterized by fluorescence probe analysis using pyrene as a probe at room temperature. Moreover, the outcomes from the conductivity and speed of sound have been corroborated with the fluorescence probe spectroscopy analysis. It would therefore be interesting to understand the structural changes and mechanism underlying the aggregation of a resultant drug–surfactant complex.