Dental fluorosis affects the quality of life. A cross-sectional, observational study was conducted in a community affected by endemic fluorosis for several generations with a conserved biological and social environment. The study included patients from the rural population of Anantapur, India. The Dean index (DI) and the Thylstrup and Fejerskov Index (TFI) were used for fluorosis classification. Additionally, water samples were collected for fluoride analysis, taken from the patients’ living areas. The statistical association between the variables was analyzed. In total, 785 patients between 10 and 60 years old were included in the study (58.7% women and 41.3% men). Fluorosis signs were found in 94.6% of patients examined using the DI and 94.4% using the TFI. Moderate–severe dental fluorosis was observed in 62.8% by DI and 73.1% by TFI consuming untreated water with up to 2.9 ppm of fluoride. Furthermore, moderate–severe dental fluorosis was observed in 33.2% by DI and 39.9% by TFI consuming water with ≤1.5 ppm of fluoride. The high prevalence of moderate–severe dental fluorosis in patients consuming water with a low fluoride concentration suggests that other factors are involved. Biological susceptibility change could play an essential role in the severity of dental fluorosis in populations exposed for several generations, affecting its actual and future quality of life.