There is an increasing concern about the co-infection of visceral leishmaniosis (VL) with human immunodeficiency virus (HIV) and/or viral hepatitis B/C. The aim of this study was to determine the prevalence of HIV and viral hepatitis co-infections among VL patients in a hyperendemic area in Eastern Sudan and to assess antibody levels in co-infected patients. This is a retrospective study where the sera of confirmed VL cases and non-VL individuals were analysed. The sera were screened for co-infections using immunochromatographic tests and ELISA for anti-HIV 1+2 antibodies, hepatitis B surface antigen (HBsAg), and anti-hepatitis C virus (HCV). Anti-Leishmania donovani antibodies in the sera of VL alone were assessed and compared to the sera of co-infected patients. Of the 100 screened VL sera, 6 (6%), 0 (0%), and 1 (1%) were positive for HBsAg, anti-HCV, and anti-HIV, respectively. These values were 5 (5%), 0 (0%), and 1 (1%) in the control group. Of note, the HCV screening test (Biorex, UK) showed positive reactivity in 32 (32%) and 17 (17%) sera of VL and control groups, respectively. All reactive sera tested negative in HCV ELISA. Of the 93 VL sera, 75 (80.6%) had strong DAT titers (1:˃102400), 2 (2.1%) demonstrated the lowest DAT titers (1:≤800), and 5 (5.4%) had marginal DAT titers (1:1600). Interestingly, the VL/HIV co-infected serum had a negative antibody titer (1:1600). Of the 6 VL/HBV co-infected sera, 1 (16.7%) and 5 (83.3%) demonstrated moderate (1:12800–1:51600) and strong (1:≥102400) DAT titers, respectively. The strong DAT titers observed in the VL/HBV co-infected sera were comparable to the DAT titers of the VL sera. The VL co-infection with HIV and hepatitis B/C is low in endemic areas in Eastern Sudan but may create a diagnostic difficulty. VL/HIV co-infected patients can have low Leishmania antibodies, thus alternative methodologies (e.g., antigen tests) may help the diagnosis.