Biomachining has been investigated as a sustainable and effective alternative to conventional prototyping techniques for molding polymeric materials for their subsequent use as microfluidic devices. A novel and simple process based on the combination of a Pressure Sensitive Adhesive mask and a varnish has been proposed for preparing metal workpieces as an alternative to photolithography, with the latter being the most widely used technique for protecting workpieces. As far as the bioprocess is concerned, it has been applied in successive mold-etching and oxidant bio-regeneration stages. Metal solubilization has proven to be repeatable in several consecutive mold-etching stages when using the regenerated oxidant solution. As a result, the lifespan of the biomachining medium has been prolonged, contributing to process sustainability. Two equations have been proposed to predict the time required to obtain a mold with a fixed height, as metal solubilization evolves differently between the first and subsequent hours. Finally, the bio-engraved copper pieces have acted as effective molds in the fabrication of self-powered polydimethylsiloxane microfluidic devices. This new biomachining application is therefore an effective and ecofriendly process for producing microfluidic devices.