Due to numerous links between trimethylamine-N-oxide (TMAO) and various disorders and diseases, this topic is very popular and is often taken up by researchers. TMAO is a low molecular weight compound that belongs to the class of amine oxides. It is formed by the process of oxidation of trimethylamine (TMA) by the hepatic flavin monooxygenases (FMO1 and FMO3). TMAO is mainly formed from nutritional substrates from the metabolism of phosphatidylcholine/choline, carnitine, betaine, dimethylglycine, and ergothioneine by intestinal microflora in the colon. Its level is determined by many factors, such as age, gender, diet, intestinal microflora composition, kidney function, and also liver flavin monooxygenase activity. Many studies report a positive relationship between the level of TMAO concentration and the development of various diseases, such as cardiovascular diseases and cardiorenal disorders, including atherosclerosis, hypertension, ischemic stroke, atrial fibrillation, heart failure, acute myocardial infarction, and chronic kidney disease, and also diabetes mellitus, metabolic syndrome, cancers (stomach, colon), as well as neurological disorders. In this review, we have summarized the current knowledge on the effects of TMAO on human health, the relationship between TMAO and intestinal microbiota, the role of TMAO in different diseases, and current analytical techniques used in TMAO determination in body fluids.