Background: CFS is a clinical state with defined symptoms, but undefined cause. The patients may show a chronic state of immune activation and treatment with an antibiotic in this subgroup has been suggested. Methods: In a retrospective study, the response of CFS patients to azithromycin, an antibiotic and immunomodulating drug, has been scored from the patients records and compared with clinical and laboratory data. Azithromycin was not the first choice therapy, but offered when the effect of counseling and L-carnitine was considered insufficient by the patient and the clinician. Results: Of the 99 patients investigated, 58 reported a decrease in the symptoms by the use of azithromycin. These responding patients had lower levels of plasma acetylcarnitine. Conclusion: The efficacy of azithromycin in the responsive patients could be explained by the modulating effect on a chronic primed state of the immune cells of the brain, or the activated peripheral immune system. Their lower acetylcarnitine levels may reflect a decreased antioxidant defense and/or an increased consumption of acetylcarnitine caused by oxidative stress.