Objectives: This study aimed to identify the emerging/reemerging pathogens in blood donation samples. Background: A metagenomic analysis has previously been used to look for pathogens but in this study, the relationship with aminotransferase (ALT) is described. Methods/materials: Excluding samples reactive to hepatitis B virus, hepatitis C virus, human immunodeficiency syndrome virus or syphilis and plasma samples were stratified into three groups of ALT levels (IU/L): A ≤ 50, B 51 to 69 and C ≥ 70, respectively. Each group was mixed in a pool of 100 samples, from which DNA and cDNA libraries were established for next generation sequencing and analysis. Pathogens of interest were identified by immunoassays, nested-polymerase chain reaction, phylogenetic analysis and pathogen detection in follow-up donors. Results: Several new or reemerging transfusion-transmitted pathogens were identified; Streptococcus suis, Babesia species and Toxoplasma gondii were found in the three ALT groups, Epstein-Barr virus (EBV) only in group C. Ten S. suis nucleic acid positive samples were detected, all closely phylogenetically related to reference strains. A donor in group A carried both S. suis genome and specific IgM in follow-up samples. This strain was identified as nontoxic S. suis. Five samples contained a short fragment of Babesia species SpeI-AvaI gene, while T. gondii was identified in 20 samples as a short fragment of 18S rDNA gene. In group C, two samples contained EBV genome. Conclusions: Blood donations that contained S. suis, Babesia species and T. gondii sequences might represent potential transfusion risks. EBV, a potential cause of elevated ALT, was detected. Metagenomic analysis might be a useful technology for monitoring blood safety.