Non-alcoholic fatty liver disease (NAFLD), most common chronic hepatic pathology, that occurs in the developed countries is estimated at 1/3 of the population. Amongst the numerous pathogenetic factors, oxidative stress and apoptosis of hepatocytes initiate many inflammatory processes and are involved in the progression of disease, particularly in transformation of non-alcoholic steatohepatitis (NASH) to cirrhosis. The aim of our study was to determine the effects of tryptophan and melatonin on the selected biochemical parameters in patients with NAFLD, and additionally, to evaluate the effects of tryptophan and melatonin in improvement of liver tissue in selected NAFLD patients. Seventy four patients with NAFLD confirmed by histopathological examination of liver biopsy samples, were admitted to the study. They were randomly assigned to three groups. Group I received the preparation Essentiale forte in the dose of 3 x 1 tablet per day and tryptophan 2 x 500 mg/day over the period of 14 months, group II received Essentiale forte and melatonin 2 x 5 mg/day over 14 months and group III received only Essentiale over the period of 14 months. In nine patients of groups I, II, and III, the liver biopsy was performed after 14-months of treatment period. Out of nine patients whom biopsy was performed, three of them were from group I, four from group II and two of them were from group III, respectively. After the 14-month treatment period, gamma-glutamyl transferase (GGPT) activity and levels of triglycerides and LDL-cholesterol were found to be significantly reduced in group I and II. The level of melatonin after the therapy was significantly elevated in group I and II and did not change in group III. Statistically significantly lower levels of IL-1, IL-6 and TNF-α were observed in patients receiving melatonin and tryptophan, comparing with group III treated with Essentiale forte only. These study findings demonstrate that melatonin and tryptophan substantially reduce the levels of pro-inflammatory cytokines and improve some parameters of fat metabolism in patients with NAFLD. In few patients with NASH melatonin and tryptophan reduced the inflammation in liver. We conclude that melatonin is worth considering for the therapy of NAFLD, particularly in patients with impaired fat metabolism accompanied by hypertriglyceridemia and hyper-LDL cholesterolemia.