This study investigated the association between dietary fatty acids (FAs) and the risk of impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM) in American adults using data from the National Health and Nutrition Examination Survey (NHANES) 2005–2016. Results showed a negative correlation between total saturated FA and IGT risk. Total FA at the fourth quintile was negatively correlated with T2DM risk compared to the first quintile. Factor analysis identified an unsaturated FA signature (including octadecenoic acid, octadecadienoic acid, octadecatrienoic acid, and eicosenoic acid) negatively associated with T2DM risk. The study suggests that several unsaturated FAs may reduce the risk of T2DM.