Tissue mechanobiology is an emerging field with the overarching goal of understanding the interplay between biophysical and biochemical responses affecting development, physiology, and disease. Changes in mechanical properties including stiffness and viscosity have been shown to describe how cells and tissues respond to mechanical cues and modify critical biological functions. To quantitatively characterize the mechanical properties of tissues at physiologically relevant conditions, atomic force microscopy (AFM) has emerged as a highly versatile biomechanical technology. In this review, we describe the fundamental principles of AFM, typical AFM modalities used for tissue mechanics, and commonly used elastic and viscoelastic contact mechanics models to characterize complex human tissues. Furthermore, we discuss the application of AFM-based mechanobiology to characterize the mechanical responses within complex human tissues to track their developmental, physiological/functional, and diseased states, including oral, hearing, and cancer-related tissues. Finally, we discuss the current outlook and challenges to further advance the field of tissue mechanobiology. Altogether, AFM-based tissue mechanobiology provides a mechanistic understanding of biological processes governing the unique functions of tissues.