Transfusion-transmissible infectious agents such as hepatitis B virus (HBV), human immunodeficiency virus (HIV), hepatitis C virus (HCV) and syphilis are among the greatest threats to blood safety for transfusion recipients and pose a serious public health problem. This cross-sectional study was undertaken with the aim of determining the seroprevalence of HIV, HCV, hepatitis B surface antigen (HBsAg) and syphilis and correlates the findings with sex and age to ascertain the associations, if any, in the occurrence of the pathogens. HBsAg, antibodies to Treponema pallidum and HCV were determined using Clinotech test strips. Antibodies to HIV types 1 and 2 were screened with Determine and Immunocomb. All the reactive samples were confirmed using enzyme-linked immunosorbent assays. Antibodies to Treponema pallidum were confirmed with a Treponema pallidum haemagglutination test. A total of 1,410 apparently healthy prospective blood donors aged between 18 and 64 years (mean+/-SD, 32.58 +/- 10.24 years) who presented for blood donation at the Ladoke Akintola University of Technology Teaching Hospital Blood Bank, Osogbo were studied. The male:female ratio was 6:1. Of the prospective blood donors, 406 (28.8%) had serological evidence of infection with at least one infectious marker and 36 (2.6%) had dual infections. The overall seroprevalence of HBsAg, HIV, HCV and syphilis was found to be 18.6%, 3.1%, 6.0% and 1.1%, respectively. The highest prevalences of HBsAg, HIV, HCV and syphilis infections occurred among commercial blood donors and those aged 18 to 47 years old, the most sexually active age group. There were no significant associations between pathogens except for syphilis and HIV (p > 0.001). The high seroprevalence of blood-borne pathogens among prospective blood donors in Osogbo, Nigeria calls for mandatory routine screening of blood donors for HBV, HIV, HCV and syphilis.