Betel nut, also known as areca nut or ‘buai’, has been highly consumed in Papua New Guinea (PNG), and more widely in South and Southeast Asia for thousands of years. This nut is intensively traded within PNG and is chewed all across the country. However, it is a matter of public health because it can spread various pathogens, through spitting, and is known to have toxic and psychoactive effects. To assess the biological impact of betel nut chewing in PNG, we analysed the human oral microbiome, a major actor of human health. Salivary samples from 271 participants (betel chewers and non-chewers) were sequenced using whole genome sequencing. We found that the oral microbiome of betel chewers is significantly less diverse than those of non-chewers. Several microbial species, some pathogenic, are enriched in the saliva of betel chewers. Our study shows that betel chewing negatively impacts the oral microbiome diversity in PNG individuals, highlighting the ongoing interaction between human biology and cultural behaviour in Papua New Guineans.