Abstract Biomethanation is an anaerobic process for converting putrescible organic matter (POM) into biogas and a digestate used for farm fertilization. The process is currently experiencing strong growth, because Québec’s waste management policy calls for a ban on POM in landfill starting in 2020. Nonetheless, there are few scientific publications on the exposure of workers involved in biomethanation operations. The general objective of this research is thus an exploratory assessment of the exposure of such workers. More specifically, this study is designed to 1) detect and measure presence and concentrations of biological agents (pathogenic and total microorganisms) and chemicals in ambient air; 2) assess possible occupational health risks; 3) rank critical areas based on potential risk. Two POM biomethanation plants participated in this project. One of the plants operates under mesophilic conditions, that is, at temperatures between 35 and 40°C, and the substrates used in this plant are wastewater sludge and POM of residential, commercial or agri-food industry origin. The other plant operates under thermophile conditions, that is, temperatures of about 55°C, and uses only POM of residential origin. Samples of ambient air were collected and analyzed to determine air concentrations of gases, metals, volatile organic compounds (VOCs), dust and bioaerosols present in the workplace. In addition, the microbial flora in the air samples were analyzed by culture and the molecular method. Because of the presence of markers of biological risk to human health, the genera Legionella and Mycobacterium and the species Saccharopolyspora rectivirgula were targeted as specific risk indicators. This study showed a biological but not a chemical risk to workers’ health related to occupational exposure to microorganisms of respirable size in concentrations that exceed recommended guideline values. In addition, there proved to be a great deal of biodiversity in the bioaerosols present. Only the non-tuberculous mycobacteria and Saccharopolyspora rectivirgula were identified as risk indicators. The bioaerosol concentrations were used to rank the most critical work areas according to the biological risks to which workers are exposed. These are the areas where POM is likely to be directly handled: the receiving and pre-treatment areas posed the greatest risk, followed by the areas used for post-treatment by aging. Nonetheless, the pre-treatment areas, where POM is stirred up by manual or mechanical mixing and spraying, remain the most critical areas, in all seasons. Given the bioaerosol concentrations in the ambient air in both biomethanation plants, workers must wear respiratory and skin protection equipment. These recommendations are especially important for those who work in proximity to or in direct contact with fresh or digested POM as well as those present during the cleaning of the equipment and the premises.