Over the last decades, bio-based composite materials have been developed as an ecological alternative to synthetic fibre-reinforced composites, and flax fibres are one of the most commonly used fibres for this purpose. The secondary cell wall (S2) and the microfibril angle (MFA) of plant fibres are the main factors responsible for the mechanical behaviour of the fibres and, consequently, for the properties of the final biocomposite material. However, the MFA values reported in the literature are obtained through heavy, time-consuming methods and often without resolution at the scale of the elementary fibres. In the present paper, for the first time, the MFAs of elementary flax fibres are measured with the alternative method of second-harmonic generation imaging under controlled polarised light (P-SHG); cotton trichomes are also investigated as a homogeneous and well-known cellulose fibre with expected contrasted MFAs compared to flax. To estimate the MFA, we analysed the images collected that clearly show the microfibrils. The values found are in line with the literature data obtained with conventional techniques. However, new important details of the microfibrils orientation of elementary flax fibres and cotton trichomes are highlighted, such as inhomogeneities in a single flax fibre, leading to MFAs varying between 0 and 10 degrees along the fibre with an average value around 5 degrees. The results obtained give an important contribution to the knowledge of the plant fibre ultrastructure, giving some structural details never provided with measurements of fibre bundles.