To further elucidate adsorption-to-partition transitional mechanisms which have been proposed previously for organo-bentonites with different surfactant loadings, the structural characteristics of interlayer surfactant aggregates on organo-bentonite with different surfactant cetyltrimethylammonium bromide loading levels (0.20–2.56 times cation exchange capacity, CEC) have been investigated by in situ X-ray diffraction (XRD) and Fourier Transform Infrared (FTIR) spectroscopy. The sorption properties and the structure of the clay interlayers changed according to the type of surfactant, the surfactant loading level, and the state of hydration in the clays. Based on the sorption of nitrobenzene, phenol, and aniline to organo-bentonites, the contaminant sorption coefficients (Ksf), normalized with the organic carbon content, show a remarkable dependence on surfactant loading levels. The Ksf values first increased with surfactant loading until reaching a maximum at 1.0 to 1.2 times the CEC, and then decreased. According to the theoretical calculation of the volume fractions relating to the interlamellar space, the interlamellar microenvironment became a more hydrophobic medium, contributing to the dissolution of organic contaminants, as the surfactant loading increased from 0.20 to 2.56 times the CEC. However, the increase in packing density (ρ) for the intercalates, and induced steric hindrances both affect the result in terms of a reduction in the accessible free space where the organic contaminants can be located, which might be a negative factor in the sorption capacity.