Background: This study aims to evaluate the percentages of voluntary and female blood donors, prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-cell lymphotropic virus (HTLV) and syphilis among blood donors and the potential association between them with ABO and Rh blood groups. Methods: A retrospective study was conducted at blood bank, King Saud Hospital, Unaizah, Qassim, Saudi Arabia for a period of 4 years (January 2013 to December 2016). After obtaining the ethical approval, 5 mL of blood each was taken from the volunteers and replacement blood donors for TTIs analysis. Results: A total of 9460 blood donor samples (9120 males (96.4%) and 340 females (3.6%)) were collected and screened. The mean age of donors was 31.4 ± 8.7 years. Seventy-six donors tested positive for TTIs (0.80%). The frequency of HBV, HCV, HIV, HTLV and syphilis were 25 (0.2%), 21 (0.22%), 1 (0.01%), 9 (0.10%) and 20 (0.21%), respectively. Most of the blood donors were found to belong to O blood group (46.45%), while AB group showed the smallest frequency rate of 5.2%. This study found no significant association between ABO and Rh blood groups with TTIs infections. Conclusion: Low percentages of voluntary and female donors were found in this study. High prevalence rates of TTIs among donors with blood group O. It is highly recommended to educate the culture and encourage the voluntary donors and enrollment of female blood donors. Also, advanced methods should be used for proper testing of donor’s blood.