The aim of the current study was to examine the factor structure and the psychometric properties of the European Portuguese version of the World Health Organization's Quality of Life Instrument in HIV Infection (WHOQOL-HIV-Bref). Exploratory factor analysis (EFA) and confirmatory factors analysis (CFA) were conducted on self-reported WHOQOL-HIV-Bref data from 1196 HIV-positive patients, frequenting the main departments of infectious diseases in Portugal. An EFA with a randomly chosen subsample suggested a five-domain structure for the WHOQOL-HIV-Bref. CFA validated this five-domain structure in a separate subsample, but also revealed that the original six-domain model fit the data. Accordingly, it was decided to use the original factor structure. Based on the original six domain structure, the European Portuguese version of WHOQOL-HIV-Bref showed acceptable internal consistency (alpha range: 0.61–0.80 across domains). All domains (with exception of Spirituality) clearly discriminated subjects considering the CD4+ T cell count subgroups. These findings revealed additional insights about the factor structure of the WHOQOL-HIV-Bref and provided evidence in support of the original six-domain structure. This instrument showed to be a cross-culturally valid measure of quality of life, particularly useful when addressing the impact of HIV not only on physical and psychological well-being but also on several domains beyond health.