Objective: To evaluate the association between circulating levels of endocannabinoids (eCBs) and non-alcoholic fatty liver disease (NAFLD). Methods: The serum levels of the main eCBs, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and their endogenous precursor and breakdown product, arachidonic acid (AA), were analyzed by liquid chromatography/tandem mass spectrometry in 105 volunteers screened for NAFLD. Hepatic ultrasound, fasting blood tests, and anthropometrics were assessed. Liver fat was quantified by the hepato-renal-ultrasound index representing the ratio between the brightness level of the liver and the kidney. Results: Patients with NAFLD had higher levels (pmol/mL) of AA (2,721 ± 1,112 vs. 2,248 ± 977, P = 0.022) and 2-AG (46.5 ± 25.8 vs. 33.5 ± 13.6, P = 0.003), but not AEA. The trend for higher levels of AA and 2-AG in the presence of NAFLD was observed in both genders and within subgroups of overweight and obesity. The association of AA and 2-AG with NAFLD was maintained with adjustment for age, gender, and BMI (OR = 1.001, 1.000–1.001 95% CI, P = 0.008 and OR = 1.05, 1.01–1.09, P = 0.006, respectively) or waist circumference. Conclusions: This study is the first to show high circulating levels of 2-AG and AA in NAFLD patients compared with controls, independent of obesity. The findings may suggest an independent role of eCBs in the pathogenesis of NAFLD.