Seroprevalence of Toxoplasma gondii has been extensively studied in a variety of different human populations. However, no study has focused on homeless populations. Accordingly, the present study aimed to assess the seroprevalence of anti-T. gondii antibodies and the risk factors associated in homeless persons from homeless shelter of São Paulo city, southeastern Brazil. In addition, anti-HIV antibodies and associated risk of T. gondii and HIV coinfection have been evaluated. Anti-T. gondii antibodies were detected by indirect fluorescent antibody test. In addition, anti-HIV levels were tested by chemiluminescence enzyme immunoassay, with positive samples confirmed by rapid immunoblot assay. Overall, IgG anti-T. gondii seropositivity was found in 43/120 (35.8%) homeless persons, with endpoint titers varying from 16 to 1,024. The only two pregnant women tested were negative for IgM by chemiluminescence enzyme immunoassay, with normal parturition and clinically healthy newborns in both cases. There were no statistical differences in the risk factors for anti-T. gondii serology (p > 0.05). Anti-HIV seropositivity was found in 2/120 (1.7%) homeless persons, confirmed as HIV-1. One HIV seropositive individual was also sero-reactive to IgG anti-T. gondii, and both were negative to IgM anti-T. gondii. This is the first study that reports the serosurvey of T. gondii in homeless persons worldwide. Despite the limited sample size available in the present study, our findings have shown that the prevalence of anti-T. gondii antibodies in homeless persons herein was lower than the general population, probably due to homeless diet habit of eating mainly processed food intake. No statistical differences were found regarding risk factors for anti-T. gondii exposure in homeless persons. Future studies should be conducted to fully establish risk factors for anti-T. gondii exposure in homeless persons.