A retrospective analysis was undertaken to determine the seroprevalence of Entamoeba histolytica infection in Sydney, Australia. Using an enzyme-linked immunosorbent assay, 429 high risk human immunodeficiency virus (HIV)-infected men who have sex with men (MSM), 446 low risk HIV-uninfected MSM, and 456 HIV-uninfected controls were assessed. Seroprevalence rates were 5.13% for the high risk HIV-infected MSM group, 0.22% for the low risk HIV-uninfected MSM group, and 0.44% for the control group. We found that high risk HIV-infected MSM have a significantly greater seroprevalence of E. histolytica with a relative risk of 22.87, when compared with low risk HIV-uninfected MSM and 11.69 when compared with controls. These findings show that in Sydney, sexually active HIV-infected MSM are at greater risk of developing amoebic disease caused by E. histolytica than HIV-uninfected MSM and the general population.