Cholesterol is one of the most important chemical substances as a structural element in human cells, and it is very susceptible to oxidation reactions that form oxysterol. Oxysterols exhibit almost the exact structure as cholesterol and a cholesterol precursor (7-dehydrocholesterol) with an additional hydroxyl, epoxy or ketone moiety. The oxidation reaction is performed via an enzymatic or non-enzymatic mechanism. The wide array of enzymatic oxysterols encountered in the human body varies in origin and function. Oxysterols establish a concentration equilibrium in human body fluids. Disease may alter the equilibrium, and oxysterols may be used as a diagnostic tool. The current review presents the possibility of using non-enzymatic oxysterols and disturbances in enzymatic oxysterol equilibrium in the human body as a potential biomarker for diagnosing and/or monitoring of the progression of various diseases.