<b><i>Introduction:</i></b> Known risk factors for multiple sclerosis (MS) include smoking, a low vitamin D status, obesity, and EBV, while the inflammatory feature of the disease strongly suggests the presence of additional infectious agents. The association between use of antibiotics and MS risk that could shed light on these factors is still undetermined. We aimed to evaluate the association between antibiotics and MS risk, in the Emilia-Romagna region (RER), Italy. <b><i>Methods:</i></b> All adult patients with MS seen at any RER MS center (2015–2017) were eligible. For each of the 877 patients included, clinical information was collected and matched to 5 controls (RER residents) (<i>n</i> = 4,205) based on age, sex, place of residence, and index year. Information on antibiotic prescription was obtained through the linkage with the RER drug prescription database. <b><i>Results:</i></b> Exposure to any antibiotic 3 years prior to the index year was associated with an increased MS risk (OR = 1.52; 95% CI = 1.29–1.79). Similar results were found for different classes. No dose-response effect was found. <b><i>Discussion/Conclusions:</i></b> Our results suggest an association between the use of antibiotics and MS risk in RER population. However, further epidemiological studies should be done with information on early life and lifestyle factors.