This study evaluated the presence of Rickettsia, Anaplasmataceae, Borrelia and Piroplasmida in Amblyomma and Ixodes spp. ticks from wild areas of Panama. Molecular detection of pathogens was performed on the following species of ticks: Amblyomma dissimile, Amblyomma mixtum, Amblyomma naponense, Amblyomma cf. oblongoguttatum, Amblyomma ovale, Amblyomma tapirellum, Haemaphysalis juxtakochi, Ixodes affinis, and Ixodes sp. nymphs. Partial sequences of Ixodes nymphs with the 16S rRNA gene showed low similarity to other Ixodes species. We found evidence of DNA from Rickettsia spp. in A. mixtum (R. amblyommatis), A. dissimile (“Candidatus R. colombianensi”), I. affinis (Rickettsia sp.), and Babesia odocolei in I. affinis. No evidence of DNA from the target microorganisms was detected in A. naponense, A. cf. oblongoguttatum, A. ovale, A. tapirellum, H. juxtakochi, or Ixodes sp. This is the first report of B. odocolei in I. affinis, which represents an additional risk of zoonosis. The detection of Rickettsia spp. corroborates previous findings in Panama. Finally, this study was crucial to determine a putative new species of Ixodes, from two nymph feeding on human.