Despite the advances that have been made in renewable energy over the past decade, crude oil or petroleum remains one of the most important energy resources to the world. Petroleum production presents many challenging issues, such as the destabilization of complex oil–water emulsions, fouling phenomena on pipelines and other facilities, and water treatment. These problems are influenced by the molecular forces at the oil/water/solid/gas interfaces involved in relevant processes. Herein, we present an overview of recent advances on probing the interfacial forces in several petroleum production processes (e.g., bitumen extraction, emulsion stabilization and destabilization, fouling and antifouling phenomena, and water treatment) by applying nanomechanical measurement technologies such as a surface forces apparatus (SFA) and an atomic force microscope (AFM). The interaction forces between bitumen and mineral solids or air bubbles in the surrounding fluid media determine the bitumen liberation and flotation efficiency in oil sands production. The stability of complex oil/water emulsions is governed by the forces between emulsion drops and particularly between interface-active species (e.g., asphaltenes). Various oil components (e.g., asphaltenes) and emulsion drops interact with different substrate surfaces (e.g., pipelines or membranes), influencing fouling phenomena, oil–water separation, and wastewater treatment. Quantifying these intermolecular and interfacial forces has advanced the mechanistic understanding of these interfacial interactions, facilitating the development of advanced materials and technologies to solve relevant challenging issues and improve petroleum production processes. Remaining challenges and suggestions on future research directions in the field are also presented.