Introduction Methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia is a common and severe disease responsible for approximately 65,000 deaths every year in Europe. Intravenous anti-staphylococcal penicillins (ASP) such as cloxacillin are the current recommended antibiotics. However, increasing reports of toxicity and recurrent stock-outs of ASP prompted healthcare providers to seek for alternative antibiotic treatment. Based on retrospective studies, cefazolin, a 1st generation cephalosporin, is recommended in patients at risk of severe ASP-associated toxicity. We hypothesized that cefazolin has a non-inferior efficacy in comparison to cloxacillin, with a better safety profile for the treatment of MSSA bacteremia. Methods and analysis The CloCeBa trial is an open-label, randomized, controlled, non-inferiority trial conducted in academic centers throughout France. Eligible patients are adults with MSSA bacteremia without intra-vascular device or suspicion of central nervous system infection. Patients will be randomized (1:1) to receive either cloxacillin or cefazolin by the intravenous route, for the first 14 days of therapy. The evaluation criteria is a composite criteria of negative blood cultures at day 5, survival, absence of relapse, and clinical success at day 90 after randomization. Secondary evaluation criteria include both efficacy and safety assessments. Three ancillary studies are planned to describe the epidemiology of β-lactamase encoding genes, the ecological impact, and pharmacokinetic/pharmacodynamic parameters of cefazolin and cloxacillin. Including 300 patients will provide 80% power to demonstrate the non-inferiority of cefazolin over cloxacillin, assuming 85% success rate with cloxacillin and taking into account loss-to-follow-up, with a 0.12 non-inferiority margin and a one-sided type I error of 0.025. Ethics and dissemination This protocol received authorization from the ethics committee Sud-Est I on November, 13th 2017 (2017-87-PP). Results will be disseminated to the scientific community through congresses and publication in peer-reviewed journals. Trial registration This trial is registered on clinicaltrial.gov (NCT03248063) and on Eudract (2017-003967-36) databases.