Abstract: This study constitutes the first contribution to the knowledge of tick dynamics and its implication in the epidemiology of rickettsial diseases in Montesinho Natural Park (MNP), Bragança district of Portugal. Of 76 ticks collected, 12 (15.8%) were Dermacentor (D.) marginatus, 36 (47.4%) D. reticulatus, and 28 (36.8%) Rhipicephalus (R.) sanguineus. Isolation assays were performed by shell-vial technique on 41 ticks. Israeli spotted fever strain was an isolate from R. sanguineus, and three isolates of Rickettsia slovaca were obtained from D. reticulatus. All 76 ticks were screened by PCR for Rickettsia sp., Ehrlichia (E.) chaffeensis, and Anaplasma (A.) phagocytophilum. Rickettsia RpA4 strain DNA was detected in 10 D. marginatus and 2 D. reticulatus, and Israeli spotted fever strain in 1 R. sanguineus. No E. chaffeensis or A. phagocytophilum infection was detected. New host records are provided for D. reticulatus. Also described for the first time in Portugal is the isolation of R. slovaca from D. reticulatus and the isolation of Israeli spotted fever strain from R. sanguineus. This confirms the association of the last rickettsiae strain with the same vector tick as previously described in Israel and Sicily.