Nanobiomedicine promises to revolutionize life quality and expectancy of patients with cognitive impairment and cancer malignancies, via unraveling key molecular processes related to their onset useful as biomarkers of disease to develop and improve the efficacy of therapies. However, it is still a challenge understanding and identifying these molecular mechanisms as biomarkers of disease, because of their high‐level of polymorphism and nanoscale dimensions. Here, it provides a review work linking the potential and capabilities of atomic force microscopy (AFM) technologies in unraveling beyond imaging the common and hidden properties of transient and nanosized molecular processes in cancer and neurodegeneration. This study highlights the most prominent operational modes of AFM to achieve morphological, mechanical, and chemical characterization of the molecular processes leading to these diseases. Finally, it outlines the advantages of AFM compared with other techniques to guide newcomers and stakeholders toward potential future avenues opened by AFM methods in nanobiomedicine.