Today, plant fibers are considered as an important new renewable resource that can compete with some synthetic fibers, such as glass, in fiber-reinforced composites. In previous works, it was noted that the pectin-enriched middle lamella (ML) is a weak point in the fiber bundles for plant fiber-reinforced composites. ML is strongly bonded to the primary walls of the cells to form a complex layer called the compound middle lamella (CML). In a composite, cracks preferentially propagate along and through this layer when a mechanical loading is applied. In this work, middle lamellae of several plant fibers of different origin (flax, hemp, jute, kenaf, nettle, and date palm leaf sheath), among the most used for composite reinforcement, are investigated by atomic force microscopy (AFM). The peak-force quantitative nanomechanical property mapping (PF-QNM) mode is used in order to estimate the indentation modulus of this layer. AFM PF-QNM confirmed its potential and suitability to mechanically characterize and compare the stiffness of small areas at the micro and nanoscale level, such as plant cell walls and middle lamellae. Our results suggest that the mean indentation modulus of ML is in the range from 6 GPa (date palm leaf sheath) to 16 GPa (hemp), depending on the plant considered. Moreover, local cell-wall layer architectures were finely evidenced and described.Molecules 2020, 25, 632 2 of 17 be found in Wambua et al. [8]. The authors studied several poly-(propylene) composites reinforced with different plant fibers (sisal, kenaf, jute, hemp, and coir), and their results showed that coir fibers, extracted from seeds, have lower longitudinal mechanical properties than the others but, on the other hand, they exhibit a higher impact strength, which was also confirmed in Reference [9]. This result follows the functional evolution of the different kinds of cells in a plant; for example, bast fibers are responsible for the stiff structure of a plant and this specific role also explains their high mechanical performance.Every bast fiber has a similar (ultra)structural model even if they originate from different plants. An elementary fiber is a single cell, and several fibers are linked to each other to form a bundle of several dozens of single fibers having a multilayer structure, as illustrated in Figure 1a: (1) the lumen is the central hollow part of the cell and its shape and diameter vary with the maturity of the plant and the environmental conditions during growth; (2) the secondary cell wall is the thickest layer, divided into two to three sub-layers (S 1 , S 2 or G, and S 3 ) rich in cellulose where the thinner and not always visible S 3 can also be assimilated to unmatured Gn layer instead of a real S 3 [10]; (3) the primary cell wall is the external layer enriched in hemicelluloses, pectins, and lignin [11]. Between the fibers, there is another layer called middle lamella (ML), which cements the primary cell walls of adjacent cells together and is mainly composed of pectic polysaccharides, lignin, and a small amou...