BackgroundClinical laboratories provide diagnostic testing services to support the effective delivery of care in today’s complex healthcare systems. Processing clinical material and the use of chemicals or radiation presents potential hazard to laboratory workers, from both biological and chemical sources. Nevertheless, the laboratory should be a safe workplace if the identification of possible hazards, clear guidelines, safety rules and infection prevention and control (IPC) precautions are applied and followed. The main aim of this systematic review was to identify, critically appraise and synthesise the research evidence to gain a clear explanation of the implementation and knowledge, attitude and practice (KAP) of IPC guidelines among hospital laboratory staff.MethodsFor this systematic review we searched MEDLINE, EMBASE, Scopus and CINAHL (EBSCO), PubMed, grey literature, reference lists and citations for studies published between database inception and November, 2021. All qualitative, quantitative and mixed-methods studies whose aim was to explore risk perception and KAP of IPC guidelines among laboratory staff in any healthcare setting were included, without language or date restrictions. Evidence was narratively synthesised into group of themes. The quality of the evidence was assessed with Joanna Briggs Institutes Critical Appraisal Tools.ResultsAfter the full-text screening, a total of 34 articles remained and were included in the final review. Thirty papers were considered to be of high quality and the remaining four were considered to be of low quality. The available evidence shows that there was good knowledge, good attitudes and moderate immunisation status, but there was still poor practice of IPC precautions and an inadequate level of training among laboratory workers.ConclusionThere is a gap among KAP related to the implementation of IPC guidelines, which indicates that laboratory staff may be at high risk of acquiring infections in the workplace. These findings suggest that training (including IPC precautions, safety policies, safety equipment and materials, safety activities, initial biohazard handling, ongoing monitoring and potential exposure) of laboratory staff to increase their knowledge about IPC precautions could improve their use of these precautions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13756-023-01257-5.