Basic knowledge of species, ecological preferences and behavior of ticks in preserved rainforests are scanty. We herein describe species richness, seasonal activity, altitude influence and questing height of ticks along 3375 m of animal trails within the largest remain of inland Atlantic rainforest in Brazil, the Iguacu National Park (INP). Altogether, 2954 ticks from nine tick species were collected; Amblyomma brasiliense Aragdo (49.09% of the specimens), Amblyomma coelebs Neumann (29.08%), Amblyomma incisum Neumann (12.49%), Haemaphysalis jwctakochi Cooley (5.21%), Amblyomma ovale Koch (2.64%), Amblyomma longirostre Koch (0.20%), Ixodes fuscipes Fonseca (0.17%), Ixodes loricatus Neumann (0.07%) and Amblyomma dubitatum Neumann (0.03%) (Ixodida: Ixodidae). There was an evident seasonal influence on tick host-questing activity, with each tick stage prevailing in a season before the following one, and a general trend of one tick generation per year. Adult tick questing height mode on vegetation showed a correspondence between tick species and preferred host size and height. Adult ticks quested higher than immatures, but several larval clusters were found high enough to seek for largesized hosts as well. Nymphal and adult tick questing height increased significantly at higher altitudes within the park. Amblyomma incisum and A. coelebs were more abundant at higher and lower alludes, respectively. The knowledge on tick species richness, ecological preferences and behavior herein obtained provides baseline information to understand tick-borne disease epidemiology occurring at landscapes under a strong anthropogenic impact