Dental fluorosis is a very prevalent endemic disease. Although oral microbiome has been reported to correlate with different oral diseases, there appears to be an absence of research recognizing any relationship between the severity of dental fluorosis and the oral microbiome. To this end, we investigated the changes in oral microbial community structure and identified bacterial species associated with moderate and severe dental fluorosis. Salivary samples of 42 individuals, assigned into Healthy (N = 9), Mild (N = 14) and Moderate/Severe (M&S, N = 19), were investigated using the V4 region of 16S rRNA gene. The oral microbial community structure based on Bray Curtis and Weighted Unifrac were significantly changed in the M&S group compared with both of Healthy and Mild. As the predominant phyla, Firmicutes and Bacteroidetes showed variation in the relative abundance among groups. The Firmicutes/Bacteroidetes (F/B) ratio was significantly higher in the M&S group. LEfSe analysis was used to identify differentially represented taxa at the species level. Several genera such as Streptococcus mitis, Gemella parahaemolysans, Lactococcus lactis, and Fusobacterium nucleatum, were significantly more abundant in patients with moderate/severe dental fluorosis, while Prevotella melaninogenica and Schaalia odontolytica were enriched in the Healthy group. In conclusion, our study indicates oral microbiome shift in patients with moderate/severe dental fluorosis. We identified several differentially represented bacterial species enriched in moderate and severe fluorosis. Findings from this study suggests that the roles of these bacteria in oral health and related diseases warrant more consideration in patients with moderate and severe fluorosis.