Background Bacterial contamination in platelet concentrates (PCs) is a major problem in transfusion medicine. Contamination with Staphylococcus aureus is occasionally missed, even with cultural screening. Study design and methods Donors implicated in S. aureus‐contaminated PC were followed up. Skin and nasal swab specimens from six donors and S. aureus isolated from PCs related to these donors were subjected to multilocus sequence typing (MLST) and pulsed‐field gel electrophoresis (PFGE) to determine the identity of bacteria. To evaluate the validity of the screening method using BacT/ALERT 3D, we spiked S. aureus and three other bacterial species as comparisons into PCs and investigated their growth pattern. Results S. aureus was isolated from all nasal specimens and from the arm skin specimens of three donors with atopic dermatitis. In all cases, the S. aureus strains isolated from the PC and those from the nasal and skin specimens of the same donor showed concordant results using MLST and PFGE. In the spiking study, S. aureus showed irregular detectability over 24 to 48 h post‐spike periods, whereas the three other bacterial species were detected in all culture bottles after a 24‐h post‐spike period. Discussion The strain identity of S. aureus between donor and PC suggests that the contaminants were derived from those colonized in the donor. Furthermore, S. aureus yielded false‐negative results using BacT/ALERT 3D.