Blood plays a major role in transmitting infectious diseases such as hepatitis C virus (HCV), human immunodeficiency virus (HIV), hepatitis B virus (HBV), syphilis, malaria, and many others. Thus, this study sought to evaluate the distribution of HCV, HIV, syphilis, and malaria among blood donors in Yemen. This is a cross-sectional study, conducted on blood donors at the national center in Yemen. Blood donors’ specimens were serologically tested for the presence of anti-HCV and anti-HIV antibodies, as well as anti-Treponema pallidum, anti-Plasmodium falciparum, and anti-Plasmodium vivax. A total of 16,367 donors were included in this study. Based on the donor’s occupation, the study showed that the relative seroprevalence of anti-HCV Ab among the donors was statistically significant, and relatively high prevalence was found among military donors (2.8%). Positive HIV antibody tests were only reported in 33 male donors (0.2%), who were mostly manual workers. A remarkably high prevalence of anti-Treponema pallidum was observed among manual workers (3.1%). There was a statistically significant difference in the distribution of anti-malaria Ab based on residency and age groups. This study revealed that the prevalence of HCV, HIV, syphilis, and malaria among donors was 2.0%, 0.2%, 2.4%, and 0.7%, respectively. Further genotyping studies are necessary to provide a complete picture of the prevalence of transfusion-transmitted infections (TTIs).