This systematic review compares polytetrafluoroethylene tape and cotton pellet when used as endodontic spacers underneath provisional restorations. The review followed the PRISMA guidelines and was registered in the PROSPERO database (CRD42020176555). Studies that compared the microbial contamination between polytetrafluoroethylene tape and cotton pellet, when used as spacers, were included. Literature searches of Pubmed, Embase, EBSCOHost Dentistry & Oral Sciences Source, Scopus, and Open Grey databases were conducted from their inception until May 2020 for studies in English or other Latin script languages. Hand searching of reference lists was performed. Three laboratory and three clinical studies were included. The risk of bias of the component studies varied widely. Results from the laboratory studies showed higher bacterial counts for cotton pellets. Results from the clinical studies showed that polytetrafluoroethylene tape was associated with a significantly lower incidence of microbial contamination. Findings were consistent throughout the studies, though the evidence available is scarce and heterogeneous. Polytetrafluoroethylene tape was associated with less microbial contamination when compared with cotton pellets as endodontic spacers and therefore appears to be a more suitable material for the purpose.