In the present study, samples of tick, tissue and blood from free-living wild small mammals (rodents and marsupials) captured in two municipalities from the Amazon biome of the Mato Grosso state, were molecularly tested for rickettsial infection. A total of 148 mammal specimens (46 rodents and 102 marsupials) were trapped, and 2,700 Amblyomma ticks (2,000 larvae and 700 nymphs) were collected. Ticks were recognized into four Amblyomma species: Amblyomma cajennense sensu stricto, Amblyomma coelebs, Amblyomma humerale, and Amblyomma scalpturatum, with new data for tick-host associations from small mammals in the Amazon biome. All tissue and blood samples of small mammals tested by PCR for rickettsial detection were negative. However, concerning tick-Rickettsia associations, Rickettsia amblyommatis was detected in all four tick species identified in the present study, being the first record for A. scalpturatum ticks. Furthermore, an uncharacterized species of Rickettsia belonging to the spotted fever group was detected in A. humerale as well.