Background: The role of opportunistic infections in accelerating disease progression in HIV-positive individuals, leading to quick death, is still receiving serious attention. The objective of this study was to determine the prevalence of Isospora belli infections in HIV-positive patients in Edo State, Nigeria between August 2007 and March 2008. Methods: A total of 268 samples from HIV-positive patients and 20 samples from HIV-negative patients were processed using the modified Ziehl-Neelsen staining technique to microscopically identify the presence of I. belli oocysts. Results: The overall prevalence of the coccidian was 3.1%. Gender and age had no correlation with the prevalence of the parasite (P > 0.05). There was a significant relationship between isosporiasis and CD4+ T cell counts in HIV-positive patients (OR=11.388, 95% CI= 2.797–46.371, P=0.0004). Conclusions: Routine investigation of I. belli in HIV-positive subjects is advocated in tertiary health institutions.