Pre-treatments for plant fibres are very popular for increasing the fineness of bundles, promoting individualisation of fibres, modifying the fibre-matrix interface or reducing water uptake. Most pre-treatments are based on the use of chemicals and raise concerns about possible harmful effects on the environment. In this study, we used physical pre-treatments without the addition of chemical products. Flax tows were subjected to ultrasound and gamma irradiation to increase the number of elementary fibres. For gamma pre-treatments, a 20% increase in the number of elementary fibres was quantified. The biochemical composition of pre-treated flax tows exhibited a partial elimination of sugars related to pectin and hemicelluloses depending on the pre-treatment. The hygroscopic behaviour showed a comparable decreasing trend for water sorption-desorption hysteresis for both types of pre-treatment. Then, non-woven composites were produced from the pre-treated tows using poly-(lactid) (PLA) as a bio-based matrix. A moderate difference between the composite mechanical properties was generally demonstrated, with a significant increase in the stress at break observed for the case of ultrasound pre-treatment. Finally, an environmental analysis was carried out and discussed to quantitatively compare the different environmental impacts of the pre-treatments for composite applications; the environmental benefit of using gamma irradiation compared to ultrasound pre-treatment was demonstrated.