Some studies have evaluated the use of Opuntia as reinforcement for polymeric matrices, obtaining good results in energy absorption tests and increasing the tensile elastic modulus. However, no studies focusing on the previous characterisation of the fibres and their treatment to improve compatibility with polymeric matrices have been found. This work analyses the chemical composition of Opuntia maxima (OM) and Opuntia dillenii (OD) cladodes and fibre, studying how different treatments influence it. AOAC 2000 methods were used to determine non-structural components and the Van Soest method was used to estimate structural components. Surface characteristics of the samples were also evaluated by Fourier Transform Infrared Spectroscopy (FTIR). Opuntia fibre presented higher cellulose (50–66%) and lignin (6–14%) content and lower hemicellulose (8–13%) content than Opuntia cladodes (9–14% cellulose, 20–50% hemicellulose, 1–4% lignin). Despite the variability of lignocellulosic materials, OD cladodes treated with water and acetic acid achieved an increase in the structural components. Alkaline fibre treatment removed pectin and hemicellulose from the fibre surface, slightly increasing the cellulose content. Future research should evaluate whether the treated Opuntia fibre can improve the mechanical properties of reinforced polymer.