Toxoplasmosis is characterized by a complex epidemiology. The risk of infection for humans depends on their contact with infective oocysts in a contaminated environment and on the amount of tissue cysts located within consumed meat. Unfortunately, the prevalence of tissue cysts is largely unknown for game species. Although herbivorous game species are a source of infection for humans, the level of infection found in wildlife can also be used to estimate environmental contamination. The aim of this study was to estimate the prevalence of Toxoplasma gondii infection and analyze its temporal dynamics in one population of chamois (Rupicapra rupicapra), one of mouflon (Ovis gmelini musimon), and two of roe deer (Capreolus capreolus) in France, surveyed during a period of 6 to 28 years. Taking into account individual risk factors, we specifically analyzed the relationship between T. gondii prevalence and meteorological conditions that may influence oocyst survival. Serum samples from 101 chamois, 143 mouflons, and 1155 roe deer were tested for antibodies against T. gondii using the modified agglutination test (MAT), an enzyme-linked immunosorbent assay (ELISA) assay, or both. Using MAT with a threshold of 1:6, seroprevalence was 14.7% in mouflon, 16.8% in chamois, and 43.7% in roe deer. In mouflon and roe deer, seroprevalence was positively correlated with age and/or body mass, in accordance with the hypothesis that antibodies have long-term persistence. In roe deer, seropositivity differed between the two populations and changed linearly over time between 1983 and 2010, increasing by a factor 1.75 every 10 years. Moreover, in this species, the highest prevalences were found during dry and cold years or during warm and moist years, depending on the population. Our results suggest that the risk for people to acquire infection through game meat increases over time, but with high variability according to the population of origin and meteorological conditions of the year.