Obtaining blood which is safe for transfusions is one of the principal challenges in the health systems of developing countries. Supply of contaminated blood increases morbidity, mortality, and the costs of patient care. In Mexico, serological screening is mandatory, but only a few of the main blood banks routinely perform a nucleic acid test (NAT). Data from 80,391 blood donations processed between August 2018 and December 2019 at the Central Blood Bank of the Western National Medical Center of the Mexican Social Security Institute (IMSS) were analyzed. All donors were screened for serological markers and NAT was performed. Reactive donors were followed-up to confirm their results. The number of reactive donors and seroprevalence rates for HIV, HCV, and HBV were 152 (18.91/10,000), 385 (47.89/10,000), and 181 (22.51/10,000), respectively; however, these rates decreased when NAT-confirmed reactive results were considered. Male donors were found to have a higher seroprevalence than females, and younger donors higher than older donors. The present study shows that HIV, HCV, and HBV seroprevalence in blood donors in Western Mexico is low. We propose that Mexico should establish future strategies, including pathogen reduction technologies (PRTs), in order to improve blood safety and reduce transfusion-transmissible infections (TTIs).