Tobacco use is known to cause health damage, partly by changing the mouth, respiratory tract, and gut-related microbiomes. This study aims to identify the associations between the human microbiome detected in domestic wastewater and the population smoking rate. Metagenomic sequencing and a biomarker discovery algorithm were employed to identify microorganisms as potential microbial biomarkers of smoking through wastewater-based epidemiology. Wastewater samples were collected from selected catchments with low and high smoking rates, i.e., 11.2 ± 1.5% and 17.0 ± 1.6%, respectively. Using the linear discriminant analysis effect size (LEfSe) method, Neisseria, Desulfovibrio, Megamonas, Blautia, Fusicatenibacter, Granulicatella and Enterococcus were suggested as potential biomarker microorganisms. A higher abundance of pathogens, including Neisseria, Eikenella and Haemophilus, was associated with the high smoking rate, likely because of their colonization in smoking-disturbed human guts. The identified potential microbial biomarkers reflect the change of the human gut microbiome due to the long-term smoking behavior. The metagenomic analysis also indicates that smoking upregulates microbial gene expression of genetic information processing, environmental information processing, and cell wall peptidoglycan cleavage, while it downregulates amino acid, lipid, and galactose metabolisms. The findings demonstrate the potential of microbial biomarkers for the surveillance of smoking through a wastewaterbased epidemiology approach.