Display omitted •The interaction between collector and graphite surface were measured by CFM and MD simulations.•Hydrophobic force dominated the interaction between collector and graphite surface.•Nonpolar collector had larger adhesion force and EDLVO force with the graphite surface.•Stronger adsorption was found between nonpolar collector and hydrophobic surface by MD simulation. Collectors are widely used in flotation to improve the interfacial hydrophobicity of valuable minerals. Clarifying the mechanism of action is therefore a prerequisite for developing new collectors for flotation intensification. In this study, chemical force microscopy was applied to directly characterize the interaction forces between a mineral surface (hydrophobic graphite) and two collector molecules (undecane and polar undecanoic acid). An undecane/undecanoic acid self-assembled monolayer was formed on the Au-coated tip of an atomic force microscopy (AFM) cantilever through chemical deposition. Jump-in phenomena were observed between the collectors and the hydrophobic surface, indicating the existence of an attractive force, which conformed to the EDLVO theories. Various experiments indicated that the interactions between undecane and the hydrophobic surface were stronger than those involving undecanoic acid, indicating that hydrophobic forces dominated the interactions. Using molecular dynamics simulations, the adsorption energies of undecane and undecanoic acid on the graphite surface were determined to be − 17.1 ± 0.3 and − 14.1 ± 0.5 kcal/mol, respectively. The consistency between the force and the adsorption energy results demonstrated that AFM is a credible technique to investigate the interaction mechanism between a mineral and a collector at a molecular level, and so will provide critical guidance for future collector screening and design.