OBJECTIVE This study aims to assess the knowledge, attitude and practice (KAP) of voluntary blood donation (BD) among the non-physician health-care workers of our institution, to investigate the relationship between these parameters and to determine the predictors of BD practice. BACKGROUND KAP surveys provide a popular context-specific evidence base for the development of blood transfusion services' strategies and interventions to promote voluntary, non-remunerated BD. However, there are just few studies worldwide assessing KAP of BD among health-care workers. METHODS This is a cross-sectional exploratory study, which took place at the 'Ippokrateio' General Hospital of Thessaloniki, Greece, a tertiary health-care institution. A specially designed, pre-tested questionnaire was distributed to all non-physician health-care workers of the hospital. A total of 1140 questionnaires were distributed. Participation in the study was optional and anonymous. Regarding data analysis, a binary logistic regression analysis was performed to describe the relationship between different elements of KAP of BD and to determine possible predictors of BD practice. RESULTS The response rate was 25·5% (291 respondents). Logistic regression analysis revealed a positive attitude towards family replacement as an important predictor of both regular and voluntary BD, with male gender also a predictor of the latter. An inconsistency between knowledge, attitude and practice of BD was revealed. CONCLUSIONS The present study adds valuable data on KAP of BD among health-care workers. This will help blood transfusion services to improve management practices among this group as they have the potential to provide a reliable, stable and safe source of blood products.