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Lyme borreliosis risk assessment process was applied in S. Rossore Estate, on the Tyrrhenian coast, near Pisa, Italy. Host-seeking Ixodes ricinus nymphs peaked in May in oak-dominated deciduous wood (median, Q1-Q3, number of nymphs/50 m dragging = 4.5, 2.5-8), whereas host-seeking larvae peaked in August in the same habitat type (6.0, 4-17/50 m dragging). Prevalence of I. ricinus infestation was 88.9% in wild rodents (n = 11), 64.3% in fallow deer (n = 28) and 0.0% in wild boars (n = 5). Borrelia burgdorferi sensu lato was not isolated from rodents’ organs, and from 80 I. ricinus nymphs and 50 adults. Moreover, PCR for B. burgdorferi sl carried out on 110 nymphs and 12 adult ticks also gave negative results. Forest workers were at higher risk of tick bite than other Estate employees (relative risk (RR): 1.7, p = 0.02). In spite of high levels of tick exposure, B. burgdorferi sl specific antibodies were not detected in sera from Estate personnel (n = 30) and sentinel animals (dogs, n = 23, fallow deer, n = 61).