Toxoplasmosis occurs worldwide and is considered one of the most important food-borne parasitic zoonoses. The consumption of undercooked meat containing viable tissue cysts and ingestion of environmental oocyst are the most important sources of infection. The aim of this retrospective study was to evaluate the spread ofToxoplasma gondiiin the province of Bologna (Emilia-Romagna region) in northern Italy, with a One Health approach, comparing seropositivity rates in different animal species and in humans over the last 19 and 4 years respectively. Analyses were performed on serological data collected over different periods at three separate locations: Istituto Zooprofilattico Sperimentale della Lombardia e della Emilia-Romagna (IZSLER); Veterinary University Hospital Clinical Pathology Service, Department of Veterinary Medical Sciences, University of Bologna; and Unit of Microbiology, St. Orsola Hospital, Bologna. Most relevant seropositivity rates observed in animals were 15.5% (wild boar), 25% (roe deer), 18.7% (goat), 29.9% (sheep), 9.7% (pigs), 42.9% and 21.8% in cat and dog, respectively. A comprehensive screening was conducted on a population of 36,814 individuals, revealing a prevalence of 20.4%. Among pregnant women, a frequence of 0.39% for active toxoplasmosis was observed. Despite certain limitations, this study provided valuable insights into the extensive distribution of this parasitic infection among diverse animal species and human populations in the province of Bologna. These findings underscore the importance of implementing consistent and proactive toxoplasmosis screening protocols during pregnancy, while emphasizing the critical need for adopting a One Health approach for effective control of this parasitic disease.