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Background: Post-donation illness can be described as appearance of clinical symptoms in blood donors after donation. The consequent call back of the donor to report these symptoms to the blood collection institution is considered a post-donation illness report (PDIR). The most suitable way to examine whether PDIR is related to infection is to apply next-generation sequencing (NGS) and viral metagenomics. Investigation into a PDIR can reveal its importance for transfusion safety and help elaborate strategies for donor education in order to prevent the transfusion transmission of infections which are not routinely tested by the blood collection services., Materials and Methods: We applied NGS and viral metagenomics on blood donations which were deferred due to a PDIR. Thirty-three PDIR donations obtained in the Blood Center of Ribeirão Preto, Southeast Brazil, were evaluated. Sequencing was performed using Illumina NextSeq 550 (Illumina Inc, San Diego, CA, USA) equipment and the reads obtained for each sample were analysed by specific bioinformatic pipeline for the classification and discovery of emerging viruses. The identified viral agents by metagenomics were directly confirmed by molecular methods., Results: In all PDIR donations, we found abundant reads of commensal viruses belonging to the Anelloviridae family as well as human pegivirus-1. However, we were also able to identify blood donations positive for clinically important viruses like dengue serotype-2 (DENV-2) of the Asian-American genotype and parvovirus B19 (B19V). Both viruses were also confirmed by real-time polymerase chain reaction, detecting DENV-2 RNA in a significant number of cases (7 samples, 21.2%), compared to B19V which was confirmed in 1 case (3.0%)., Discussion: Our study applies for the first time viral metagenomics to evaluate the significance of PDIRs. We confirm the crucial importance of the donor providing a timely PDIR for the prevention of transfusion transmission of viral infections which are not routinely tested in the blood banks worldwide.