The upper part of the Santa Rosita Formation (Ordovician, Tremadocian) in the Nazareno area, Cordillera Oriental, northwestern Argentina, records the vertical passage of high-energy, shallow water platform environments to offshore settings. Eighteen trilobite species are described from this locality for the first time. Although the taxa from the lower part of the succession (Leptoplastides sp., Asaphellus sp.) are scarce and non-age diagnostic, those from the upper part include diverse assemblages partially assigned to the late Tremadocian Notopeltis orthometopa Zone. Systematic descriptions of several species (Geragnostus nesossii Harrington and Leanza, G. callaveiformis Harrington and Leanza, Asaphellus jujuanus Harrington, Notopeltis orthometopa [Harrington], Mekynophrys nanna Harrington, Ceratopyge forficuloides Harrington and Leanza, Apatokephalus tibicen Přibyl and Vanĕk) are improved, the genus Nileus Dalman (including N. australis n. sp.) is first reported from the Tremadocian of western Gondwana, and new species of Asaphellus Callaway (A. nazarenensis n. sp.), Conophrys Callaway, and Apatokephalus Brøgger are described. The trilobites have their closest affinities with faunas from Norway and Sweden. Notopeltis orthometopa and Mekynophrys nanna are restricted to the uppermost part of the succession, well above the first records of most other trilobites recognized.