•Microstructure of nopal spines was characterized by microscopy techniques.•Distribution of cellulose and lignin in nopal spines was evidenced by microscopy.•Micromechanical properties are influenced by the structural arrangement of spines.•Nopal spines are harder than wood materials.•Nopal spines could be an alternative source of cellulose and lignin. The aim of this work was to study the microstructure and micromechanical properties of spines obtained from nopal waste. Spines were obtained by drying at 40 °C then sieving. Physical and chemical assays and microscopy techniques were used to determine the role of microstructural arrangement in local micromechanical properties. Transversal (TS) and longitudinal sections (LS) of the spines were studied by indentation and microscopy. Environmental scanning electron microscopy was helpful for characterizing the overall structure of spines. Confocal laser scanning microscopy was used for determining the distribution of cellulose and lignin in spines, which was associated with their micromechanical properties. Atomic force microscopy showed that TS is less rough (Ra = 3.08 ± 0.75 nm) and more hard (0.57 ± 0.31 GPa) than LS (Ra = 24.56 ± 1.60 nm, 0.57 ± 0.31 GPa). In accordance with these results, the elastic modulus of LS (8.65 ± 3.18 GPa) is lower than that of TS (14.94 ± 7.09 GPa). The hardness and elastic modulus of libriform fibers and sclerified epidermis are influenced by their microstructures and as well as the distribution of cellulose and lignin in the spines. The microstructural arrangement and the distribution of cellulose and lignin in the TS provide greater hardness values than those of woods. The current study presents a novel structural characterization of nopal spines and their local micromechanical properties. This waste product could be a cheap and non-wood alternative resource of cellulose with good mechanical properties useful for designing novel biomaterials with applications in the agricultural sector.