Background Mitoxantrone (MX) has been used as second line therapy for aggressive multiple sclerosis (MS). Potential cardiotoxic effects of MX limit its use; a cumulative dose of up to 100 mg/m2, has been long considered relatively safe. We calculated the frequency of cardiac side effects in MS patients treated with MX. Methods We performed a cohort study including all MS patients treated with MX at the Neurological Department of the University Hospital of Palermo, Italy. Two hundred-sixty-four MS patients diagnosed according to validated criteria were included and followed-up until the end of September 2010. Patients were treated with MX as a second line therapy if they had no previous heart diseases determined by clinical evaluation, electrocardiography, and echocardiography. Treatment administration was made at a monthly dose of 8 mg/m2 for the first three months and at a dose of 12 mg/m2 every three months. Echocardiography was routinely performed every six months. Treatment was stopped before reaching the final dose if signs had appeared of impaired heart function, confirmed left ventricular ejection fraction reduction lower to 50%, or a confirmed reduction of more than 10% with respect to the first examination. Results Heart involvement was observed in 12.4% of treated individuals, and symptomatic congestive heart failure occurred in 2.7% of the cohort. A patient had a myocardial infarction, and 3.1% showed electrocardiographic anomalies not present at baseline. Conclusion Our study confirms that cardiac adverse events associated with MX are more common than previously reported.