Dodecylamine (DDA) and sodium oleate (OL) are commonly used collectors in the reverse flotation and the direct flotation of goethite. However, the flotation mechanisms of DDA and OL on the goethite surface remain unclear. In this study, the first-principles density functional theory calculations were used to reveal the role of the hydration of the goethite surface and its effects on flotation reagents from a microscopic perspective. The calculation results showed that DDA was adsorbed on the surface of goethite by hydrogen bonds in the absence of hydration. However, the existence of the hydration microstructure hindered the formation of hydrogen bonds and made it difficult for DDA to be adsorbed on the goethite surface. In the OL system, oleate ions are chemically adsorbed on the surface Fe sites of goethite in the absence of hydration, while in the presence of hydration, the oleate ions were adsorbed on the H-terminal hydration surface of goethite by hydrogen bonds. This work sheds new light on the roles of the hydration microstructure and the adsorption mechanism of the flotation reagent on the oxide minerals.