Plant cell walls are highly dynamic, complex structures composed of multiple biopolymers that form a scaffold surrounding the plant cell. A nanoscale understanding of their architecture, mechanical properties, and formation/degradation dynamics is crucial for revealing structure-function relationships, mechanisms of shape formation, and cell development. Although imaging techniques have been extensively used in recent decades to reveal the structural organization and chemical compositions of cell walls, observing the detailed native architecture and identifying the physicochemical properties of plant cell walls remains challenging. Atomic force microscopy (AFM) is a powerful tool for simultaneously characterizing the morphology, nanomechanical properties, single-molecule interactions, and surface potentials of living biological systems. However, studies employing AFM to investigate plant cell walls have been relatively scarce. In this review, we discuss the latest advancements in AFM for in situ imaging of the multidimensional structure of the cell wall, measuring the mechanical properties of plant tissues or single cells, specific single-molecule recognition of cell wall-related enzymes-polysaccharides, and detecting the Kelvin potential of plant cell walls. We emphasize the fundamental challenges of AFM in characterizing plant cell walls and review potential applications for state-of-the-art AFM-based infrared/Raman spectroscopy toward answering open questions in plant biology.