The orientation of cellulose microfibrils within plant fibres is one of the main factors influencing their mechanical properties. As plant fibres are more and more used as reinforcement for agro-composites, their mechanical properties have a strong influence on the final composite properties. It is, therefore, of interest to obtain reliable information about the microfibril angle (MFA) to better support the choice of fibres depending on the product requirements. In the present study, the reliability and specificities of three non-destructive methods that allow analysis on the same fibre glued on a holder; X-ray diffraction (XRD), second harmonic generation (SHG) and transmission ellipsometry (TE) microscopy; are investigated. Three types of plant fibres, with both low (nettle), and high (cotton, sisal) MFA values, are compared and their geometry and biochemical composition are characterised. The results obtained on the same fibre confirm that MFA analysis remains tedious and that despite their limitations, the methods are complementary depending on the information requested. Indeed, SHG is recommended for direct, qualitative and plane-selective mapping of heterogeneities in macrofibril orientations at various depths. However, reliable quantitative results with SHG depend on the initial image quality and could benefit from further image processing refinement. On the contrary, XRD and TE measure MFAs over the entire fibre thickness and provide variations along the fibres if a sufficient optical/spatial resolution is reached. Regarding the characterization of intrinsic defects in plant fibres, both SHG and TE suffer from uncertainties induced by the disorganization of the microfibril network and the lack of symmetry between the front and back fibre walls. Finally, all techniques prove to be dependant on the initial fibre alignment and geometry (i.e. twisting, double fibre configuration or form factor) which vary along the fibre length and should be carefully taken into account.