Objectives The increased demand for animal blood transfusions creates the need for an adequate number of donors. At the same time, a high level of blood safety must be guaranteed and different guidelines (GLs) deal with this topic. The aim of this study was to evaluate the appropriateness of different GLs in preventing transfusion-transmissible infections (TTI) in Italian feline blood donors. Methods Blood samples were collected from 31 cats enrolled as blood donors by the owners' voluntary choice over a period of approximately 1 year. Possible risk factors for TTI were recorded. Based on Italian, European and American GLs, specific TTI, including haemoplasmas, feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), Anaplasma phagocytophilum, Ehrlichia species, Bartonella species, Babesia species, Theileria species, Cytauxzoon species, Leishmania donovani sensu lato and feline coronavirus (FCoV), were screened. Rapid antigen and serological tests and biomolecular investigations (PCR) were used. Several PCR protocols for haemoplasma and FeLV DNA were compared. Results The presence of at least one recognised risk factor for TTI was reported in all cats. Results for FIV and FeLV infections were negative using rapid tests, whereas five (16.1%) cats were positive for FCoV antibodies. Four (12.9%) cats were PCR positive for haemoplasma DNA and one (3.2%) for FeLV provirus, the latter being positive only using the most sensitive PCR protocol applied. Other TTI were not detected using PCR. Conclusions and relevance Blood safety increases by combining the recommendations of different GLs. To reduce the risk of TTI, sensitive tests are needed and the choice of the best protocol is a critical step in improving blood safety. The cost and time of the screening procedures may be reduced if appropriate tests are selected. To this end, the GLs should include appropriate recruitment protocols and questionnaire-based risk profiles to identify suitable donors.