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A prior investigation in 1993 identified a high seroprevalence of toxoplasmosis (63%) in the Canary Islands. This study aims to assess the current prevalence of the disease in diverse population groups. The study was based on a population-scale screening involving 273 residents utilizing <i>T. gondii</i> IgG ELISA and a 20 year retrospective study (1998–2018). This included AIDS/HIV outpatients (1357, of which 324 were residents), AIDS/HIV hospitalized patients (741) and patients with fever of intermediate duration (158). The seroprevalence in the resident population was 37%, with significant differences between islands. Among resident outpatients with AIDS/HIV, 14.2% had specific anti-<i>T. gondii</i> IgG, and three had anti-<i>T. gondii</i> IgM; however, IgG avidity testing indicated non-active infection. In patients hospitalized for AIDS/HIV, <i>T. gondii</i> causing encephalitis was detected in 2%. Among patients with fever of intermediate duration, 28.5% were positive for <i>T. gondii</i> IgG, and four also showed IgM positivity, although the infection was non-active. The study reveals a decrease in human toxoplasmosis over the past 30 years. However, the current seroprevalence, which stands at 37%, together with the substantial risk that <i>T. gondii</i> represents for immunocompromised individuals, highlights the need to implement preventive and control strategies to control the threat that this infection can pose to public health in the Canary Islands population.