Background: Genital ulcer disease (GUD) is a sexually transmitted disease characterised by ulcerating lesions. Despite the introduction of sexually transmitted infections (STIs) syndromic management approach into primary healthcare in South Africa (SA) in 1995, the prevalence of STIs in South Africa remains high.Objectives: The study investigated the aetiology of GUD and factors influencing it among public community health centre (CHC) attendees in the Eastern Cape, South Africa.Method: A total of 105 participants were recruited among individuals presenting with GUD from three CHCs located in the Eastern Cape Province, South Africa. Blood and genital ulcer samples were collected from consented participants. Blood samples with suitable sera were tested for human immunodeficiency virus (HIV) and syphilis. Herpes simplex virus types 1/2 (HSV–1/2), Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi and Klebsiella granulomatis were detected in nucleic acid extracted from genital ulcer specimens.Results: Out of the 98 samples with suitable sera, 55.1% and 8.2% were HIV and syphilis seropositive, respectively. Ulcerating STI pathogens were detected in 31.4% of the study participants. Herpes simplex virus type 2 was the most detected pathogen (16.2%) followed by Chlamydia trachomatis (10.5%), HSV-1 (8.6%), Haemophilus ducreyi (8.6%) and Treponema pallidum (6.7%). Multiple pathogens were detected in 13.3% of participants. Detected multiple ulcerating pathogens were common among HIV-positives (p = 0.016).Conclusion: Molecular methods for diagnosing pathogens have the potential to improve the management of GUD. Data generated from this study would contribute to the limited data on GUD in the Eastern Cape Province. Further research with a larger sample size is recommended.Contribution: Data generated would contribute to the limited data on GUD in the Eastern Cape province, South Africa.