Blood donation is vital for healthcare; however, transfusion-transmitted infections (TTIs) pose a serious risk. This study investigated the seroprevalence of TTIs among Saudi blood donors. This retrospective study included male blood donors aged ≥ 18 years who donated blood at Al-Noor Specialist Hospital in Makkah from January 2017 to December 2022. The blood units were screened for hepatitis B surface antigen (HBsAg) and core antibodies (HBc-IgG), hepatitis C antibodies (HCV-Abs), syphilis, HIV-1 antigen/antibody (HIV-1 Ag/Ab), human T-lymphotropic virus 1, 2 (HTLV-1/2), and malaria. There were 40,287 donors with an average age of 44.33 ± 18.12 years, and 62.3% (n = 25103) were Saudis. The overall rate of TTIs seropositivity was 7.4% (n = 2953); HBc-IgG (6.1%; n = 2473) was the most common, followed by HCV-Abs (0.4%; n = 177), and syphilis (0.34%; n = 136). All cases were negative for malaria, whilst HIV and HTLV positive donors were 0.06% (n = 24) and 0.13% (n = 52), respectively. Syphilis was more prevalent among non-Saudis (0.24%; n = 83) than among Saudis (0.1%; n = 53), whereas anti-HBc antibodies seropositivity was significantly higher among Saudi (3.4%; n = 1373) than non-Saudi donors (2.7%; n = 1100). Hepatitis B virus was the most frequently detected bloodborne pathogen, followed by hepatitis C virus and syphilis. Hepatitis B virus was also more prevalent among Saudi donors, whilst expatriates had higher rates of syphilis. Additional prospective multicenter studies are needed to accurately determine the prevalence of TTIs in Saudi Arabia.