Optimizing material removal rate has been one challenge in developing biomachining, an alternative option for the green machining process for metal substrates. As such, several research papers have tried to seek new knowledge by adjusting biomachining parameters such as temperature, shaking rate, pH, and Fe+ concentration. This research provides new knowledge to optimize the biomachining material removal rate by adding oxygen to the bacteria’s culture. Oxygen is an important factor in the aerobic metabolism of Acidithiobacillus ferrooxidans, the biomachining agent. An air supply system adds oxygen to the bacteria’s culture. The added oxygen successfully enhances the material removal rate of the biomachining process by an average enhancement of about four times through a copper biomachining process. On the other hand, an increment of surface roughness of about two times was also observed. These effects of oxygen are important findings to overcome the low material removal rate of the biomachining. On a further note, to optimize the biomachining process, this process should be combined with other known parameters such as temperature, shaking rate, pH, and Fe+ concentration. An optimized process of biomachining will have a wide range of material removal rates and surface roughness. It can be utilized in MEMS fabrications such as micro/nano copper antennas, micro heat exchangers, and lab-on-chip devices. © 2023, The Author(s), under exclusive licence to Korean Society for Precision Engineering.