Despite the development of biorefinery processes, the possibility of coupling the “conventional” composting process with the production of biochemicals is not taken into account. However, net carbon monoxide (CO) production has been observed during bio-waste composting. So far, O2concentration and temperature have been identified as the main variables influencing CO formation. This study aimed to investigate CO net production during bio-waste composting under controlled laboratory conditions by varying aeration rates and temperatures. A series of composting processes was carried out in conditions ranging from mesophilic to thermophilic (T = 35, 45, 55, and 65 °C) and an aeration rate of 2.7, 3.4, 4.8, and 7.8 L·h−1. Based on the findings of this study, suggestions for the improvement of CO production throughout the composting process have been developed for the first time. The highest concentrations of CO in each thermal variant was achieved with an O2deficit (aeration rate 2.7 L·h−1); additionally, CO levels increased with temperature, reaching ~300 ppm at 65 °C. The production of CO in mesophilic and thermophilic conditions draws attention to biological CO formation by microorganisms capable of producing the CODH enzyme. Further research on CO production efficiency in these thermal ranges is necessary with the characterization of the microbial community and analysis of the ability of the identified bacteria to produce the CODH enzyme and convert CO from CO2.