Given its detrimental consequences, biofouling is a central problem in industrial applications that affects submerged artificial abiotic surfaces. For instance, it shortens the service life of surfaces operating in marine and industrial environments, such as ship hulls, pipelines, heat exchangers, water filtration membranes, biosensors, and medical implants and concomitantly increases maintenance demands. In recent years, research has been propelled by the urge to minimize the replacement and maintenance costs related to the biofouling of abiotic surfaces. Colloidal probe atomic force microscopy (AFM) is a crucial analytical technique for quantifying interfacial interactions and unveiling the underlying biofouling mechanisms for a broad range of substrates. This technique is used to measure surface and colloidal forces by retracting individual colloidal particles immobilized at the apex of the AFM cantilever away from a planar surface under the conditions of choice. These forces are believed to dominate the biofouling behavior of surfaces. Several studies have utilized the colloidal probe AFM technique to estimate the adhesion of biofoulants to surfaces and elucidate the various aspects governing this process. This review summarizes the applications of colloidal probe AFM, including but not limited to characterizing the properties of abiotic surfaces, such as adhesive interactions, mechanical properties, and biofouling propensities. In addition, recent applications of colloidal probes relevant to biofouling are outlined. Furthermore, studies using various modified colloidal probes to test the biofouling resistance/anti-biofouling capacity of modulated abiotic surfaces are included.