BACKGROUND: Co-infection with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) may lead to accelerated hepatic disease progression with higher rates of liver cirrhosis and liver-related mortality compared with HBV mono-infection. Co or super-infection with hepatitis Delta virus (HDV) may worsen the liver disease and complicate treatment possibilities. METHODS: In this cross-sectional study we included HIV-infected individuals who had a routine blood analysis performed at an HIV clinic in Bissau, Guinea-Bissau between the 28th of April and 30th of September 2011. All patients were interviewed, had a clinical exam performed and had a blood sample stored. The patients' samples were tested for HBV and HDV serology, and HBV/HDV viral loads were analyzed using in-house real-time PCR methods. RESULTS: In total, 576 patients (417 HIV-1, 104 HIV-2 and 55 HIV-1/2) were included in this study. Ninety-four (16.3%) patients were HBsAg positive of whom 16 (17.0%) were HBeAg positive. In multivariable logistic regression analysis, CD4 cell count <200 cells/µl and animist religion were significantly associated with HBsAg positivity. Due to scarcity of available plasma, virological analyses were not performed for eight patients. HBV DNA was detected in 42 of 86 samples (48.8%) positive for HBsAg and genotyping was performed in 26 patients; 25 of whom had genotype E and one genotype D. Among 9 patients on antiretroviral treatment (ART), one patient had the [L180M, M204V] mutation associated with lamivudine resistance. Among the HBsAg positive patients 25.0% were also positive for anti-HDV and 4/9 (44.4%) had detectable HDV RNA. CONCLUSION: HBV and HDV were frequent co-infections among HIV positive patients in Guinea-Bissau and chronic infection was associated with severe immunosuppression. Lamivudine was widely used among HBsAg positive patients with the risk of developing resistant HBV.