Piezoelectric polymers are capable of interconverting mechanical and electrical energy, and are therefore candidate materials for biomedical applications such as sensors, actuators, and energy harvesters. In particular, nanowires of these materials are attractive as they can be unclamped, flexible and sensitive to small vibrations. Poly-l-lactic acid (PLLA) nanowires have been investigated for their use in biological applications, but their piezoelectric properties have never been fully characterised, even though macroscopic films and fibres have been shown to exhibit shear piezoelectricity. This piezoelectric mode is particularly interesting for in vivo applications where shear forces are especially relevant, and is similar to what has been observed in natural materials such as bone and DNA. Here, using piezo-response force microscopy (PFM), we report the first direct observation of shear piezoelectricity in highly crystalline and oriented PLLA nanowires grown by a novel template-wetting method. Our results are validated using finite-element simulations and numerical analysis, which importantly and more generally allow for accurate interpretation of PFM signals in soft nanostructured materials. Our work opens up the possibility for the development of biocompatible and sustainable piezoelectric nanogenerators and sensors based on polymer nanowires.