Background: Yemen is facing major challenges in ensuring the safety and availability of blood transfusion to meet the increased demand for blood transfusion due to the protracted conflict. However, Transfusion-transmissible infectious (TTIs) agents such as hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), syphilis and malaria remain the greatest threats for blood transfusion safety in such fragile, conflict-affected and vulnerable settings. This study aimed to determine the magnitude of TTIs among blood donors attending National Blood Transfusion and Research Center (NBTRC) and its associated factors. Methods: Cross- sectional study was conducted on 340 blood donors at the NBTRC during November and December 2017. Data was collected through face-to-face interview using a predesigned questionnaire that covered socio-demographic characteristics and possible TTIs associated factors. Blood samples were drawn and tested for HBV surface antigen (HBsAg), HCV antibodies, HIV1, 2 by using electrochemi-luminescence immunoassay technique and rapid immunochromato-graphic for screened syphilis and malaria antibodies. Result: The overall prevalence of TTIs was 8.8% where 2.5 %, 1.2%, 0.3%, 1.2%, 3.2% have HBV, HCV, HIV, syphilis and malaria respectively. HBV was significantly associated with history of jaundice and cupping. Furthermore, urethero-vaginal excretion was significantly associated with syphilis while malaria was significantly higher among donors from malaria endemic areas. Nearly three-fourth of donations were from replacement donors who had significantly higher TTIs prevalence than the voluntary donors: 10.4% vs. 3.3%, OR 3.4 (CI: 1.1-11.6). Conclusions: Although the prevalence of TTIs is low, it poses a serious risk for blood recipients especially in in fragile, conflict-affected and vulnerable settings where needs for blood transfusion are increasing and resources are limited. Therefore, using more sensitive screening methods and establishment of TTIs surveillance system should be considered. Efforts should be made to improve donor recruitment procedures and increase the proportion of regular and voluntary donation.