The purpose of this study was to improve our understanding of the molecular organization of hydrophobic guest molecules in the presence of co-adsorbed water inside SBA-15 ordered mesoporous silica material. Understanding this adsorption competition is essential in the development of applications of controlled adsorption and desorption. The poorly water soluble drug compound itraconazole and the fluorescent probe Nile red were selected for the study. The interaction between itraconazole and SBA-15 was investigated using FT-IR, (1)H MAS NMR and (29)Si MAS NMR spectroscopy, by determination of adsorption isotherms and release kinetics in simulated gastric fluid. The distribution and migration of the hydrophobic fluorescent probe Nile red was visualized in situ using confocal fluorescence microscopy. For both molecules, there was a pronounced influence of the co-adsorbed water on adsorption, hydrophobic aggregation and migration in SBA-15 pores. These insights contribute to the development of practical methods for loading ordered mesoporous silica materials with hydrophobic molecules.