Accreditation of medical laboratories in Croatia is carried out by the Croatian Accreditation Agency according to the international standard ISO 15189. In Croatia, accreditation is carried out on a voluntary basis and only 8 laboratories are currently accredited for clinical chemistry and laboratory hematology testing. The aim of this study was to investigate the attitudes of laboratory staff of the University Hospital Centre Zagreb on laboratory accreditation, to examine the perceived advantages and disadvantages of accreditation and to determine whether there are differences in staff attitudes towards accreditation and their familiarity with accreditation requirements depending on education level and age. The survey was conducted prior to the annual accreditation review at the end of 2020, partly using the online survey software SurveyMonkey and partly in paper form. The survey questionnaire contained 34 questions; the first part of the questionnaire referred to the demographic characteristics of the respondents, and the second part of the survey contained their views on accreditation and the quality system. A total of 118 responses were collected. Most respondents have a positive attitude towards accreditation and prefer to work in an accredited laboratory. They cite better documentation of work processes as the main advantage of accreditation, while on the other hand they cite too much documentation as the main disadvantage. Most respondents believe that accreditation has increased their workload, but they consider keeping accreditation documentation to be appropriately demanding. Annual accreditation inspections are perceived by staff as additional stress. Respondents with a master’s degree (at least) are more likely to have a positive attitude towards accreditation, more often believe that accreditation achieves increased reliability of results and are better acquainted with the requirements of ISO 15189 compared to those who completed secondary school and those with a bachelor’s degree. Therefore, a continuous training program should be provided for the latter and their suggestions for improving the quality system should be taken into account in order to ensure adherence to the prescribed protocols, improve motivation and use the full potential of all staff. The age of the respondents does not affect their attitudes about accreditation. This is the first study to examine the attitudes of laboratory staff in Croatia towards accreditation, and the analysis of the results revealed new insights that can be applied to continue to improve the quality of laboratory services and improve the quality assurance system.