Background/Objectives: Transfusion-transmitted infections (TTIs) remain an important concern in blood safety. Nucleic acid amplification testing (NAT) shortens the diagnostic window period relative to conventional serological assays. This study aimed to determine the prevalence of Hepatitis B virus(HBV), Hepatitis C virus (HCV), and Human immunodeficiency virus (HIV) among blood donors and to evaluate concordance between NAT and conventional serological screening methods. Methods: A retrospective cross-sectional study was conducted at Riyadh Regional Laboratory from May 2021 to May 2022 and included 48,901 blood donors. Donor samples were tested for HBV, HCV, and HIV using serological assays (chemiluminescent immunoassays on the Abbott Alinity s system) and NAT. Data were analyzed using SPSS. Results: The prevalence of HCV was 0.149% by serological testing and 0.061% by NAT. HBV prevalence was 0.252% by serology and 0.264% by NAT, while HIV prevalence was 0.041% and 0.037%, respectively. NAT identified 25 seronegative but NAT-reactive donations, including 13 HBV, 11 HCV, and 1 HIV donation. A statistically significant difference between NAT and serology was observed for HCV (p < 0.001, McNemar’s test), but not for HBV (p = 0.180) or HIV (p = 0.317). Conclusions: The prevalence of major viral TTIs among blood donors was low, with HBV being the most frequently detected infectious agent. NAT and serological assays provide complementary information and support the continued use of combined screening strategies to enhance transfusion safety.