Polylactic acid (PLA) has become one of the most commonly used polymers in medical devices given its biocompatible, biodegradable and bioabsorbable properties. In addition, due to PLA’s thermoplastic behaviour, these medical devices are now obtained using 3D printing technologies. Once obtained, the 3D-printed PLA devices undergo different sterilisation procedures, which are essential to prevent infections. This work was an in-depth study of the physicochemical changes caused by novel and conventional sterilisation techniques on 3D-printed PLA and their impact on the biological response in terms of toxicity. The 3D-printed PLA physicochemical (XPS, FTIR, DSC, XRD) and mechanical properties as well as the hydrophilic degree were evaluated after sterilisation using saturated steam (SS), low temperature steam with formaldehyde (LTSF), gamma irradiation (GR), hydrogen peroxide gas plasma (HPGP) and CO2 under critical conditions (SCCO). The biological response was tested in vitro (fibroblasts NCTC-929) and in vivo (embryos and larvae wild-type zebrafish Danio rerio). The results indicated that after GR sterilisation, PLA preserved the O:C ratio and the semi-crystalline structure. Significant changes in the polymer surface were found after HPGP, LTSF and SS sterilisations, with a decrease in the O:C ratio. Moreover, the FTIR, DSC and XRD analysis revealed PLA crystallisation after SS sterilisation, with a 52.9% increase in the crystallinity index. This structural change was also reflected in the mechanical properties and wettability. An increase in crystallinity was also observed after SCCO and LTSF sterilisations, although to a lesser extent. Despite these changes, the biological evaluation revealed that none of the techniques were shown to promote the release of toxic compounds or PLA modifications with toxicity effects. GR sterilisation was concluded as the least reactive technique with good perspectives in the biological response, not only at the level of toxicity but at all levels, since the 3D-printed PLA remained almost unaltered.