BackgroundArchitectural division of aseptic and septic operating theatres is a distinct structural feature of surgical departments in Germany. Internationally, hygienists and microbiologists mainly recommend functional separation (i.e. aseptic procedures first) without calling for separate operating floors and rooms. However, patients with severe musculoskeletal infections (e.g. joint empyema, spondylodiscitis, deep implant-associated infections) may benefit from the permanent availability of septic operating capacities without delay caused by an ongoing aseptic surgical program. A systematic literature review on the influence of a structural separation of septic and aseptic operating theatres on process and/or outcome quality has not yet been conducted.MethodsSystematic literature search in PubMed MEDLINE, Ovid Embase, CINAHL and the Cochrane Library, screening of referenced citations, and assessment of grey literature.ResultsA total of 572 articles were found through the systematic literature search. No head-to-head studies (neither randomised, quasi-randomised nor observational) were identified which examined the impact of structural separation of septic and aseptic operating theatres on process and/or outcome quality.ConclusionsThis review did not identify evidence in favour nor against architectural separation of septic or aseptic operating theatre. Specifically, there is no evidence of a harmful effect of architectural separation. Unless prospective studies, ideally randomised trials, will be available, it is unjustified to call for abolishing established hospital structures. Future investigations must address patient-centered endpoints, surgical site infections, process quality and hospital economy.Systematic review registrationPROSPERO (International prospective register of systematic reviews): CRD42018086568.Electronic supplementary materialThe online version of this article (10.1186/s13643-018-0937-9) contains supplementary material, which is available to authorized users.