Non-alcoholic fatty liver disease (NAFLD) and Alzheimer’s disease (AD) are common co-morbidities in patients with diabetes mellitus type 2 (T2DM). Resistance to insulin constitutes a major etiological factor for both conditions as well as in T2DM; thus, hypoglycemic drugs may show some promising prospect in this category of patients. Additionally, cholinesterases (ChEs), mainly butyrylcholinesterase (BChE) are, etiologically, a factor in both AD and DM; also, the effect of antidiabetic drugs on enzyme activity is a reasonable target for response. The present work aimed to investigate the effects of metformin and glibenclamide (MET-SU) with regard to acetylcholinesterase (AChE) and BChE activities and liver biochemical profile in 2 T2DM patient groups: those with a clinical picture of NAFLD and those free from NAFLD. The study is a case-control study, involving 3 groups (30 subjects each). Group 1 comprised of T2DM patients free from clinically evident NAFLD and group 2 comprised of T2DM patients newly diagnosed with NAFLD. The control group consisted of apparently healthy subjects. Groups 1 and 2 were on a metformin-glibenclamide (MET-SU) combination. AChE and BChE activities, as well as levels of transaminases, serum albumin, total bilirubin, alkaline phosphatase and fasting glucose serum levels were tested. AChE and BChE activity in groups 1 and 2 were significantly different compared to control, but much lower in group 2 compared to group 1 and control. In comparison to controls, plasma levels of transaminases, fasting glucose and alkaline phosphatase in group 2 showed a significant increase, while other parameters were not significantly different. We conclude that diabetic patients with NAFLD who are treated with a metformin-glibenclamide combination showed a better cholinesterase enzyme profile but less favorable liver function panel compared to other groups. These findings suggest a potential protective effect of the drugs in diabetic-NAFLD patients manifested as lowered cholinesterase activities, but they also indicate an apparent worsening of liver condition, in terms of biochemical parameters of liver function.