Bioapatite of fossil bone and teeth is susceptible to alteration and ion exchange during burial and diagenesis, varying its Sr content through the geological time. Nevertheless, fossil shark teeth are a powerful proxy for both chronostratigraphic and paleoecological reconstructions, thanks to the presence of the enameloid, a hard outer layer consisting of resistant fluorapatite crystallites. Here, we analyze fossil shark teeth from the Miocene sediments of the Chilcatay Formation of the Pisco Basin (southwestern Peru) with the aim of dating poorly constrained strata in this region. (Ultra)structural and compositional analyses of fossil lamniform and carcharhiniform teeth are performed through macroscopical observations, optical microscopy and SEM-EDS for evaluating the preservation state of the collected teeth. Shark teeth display a compact and well preserved outer enameloid layer formed by highly ordered bundles of crystallites that is distinctly separated by a more porous and heterogeneous inner core of dentine featuring diagenetic artefacts and microborings. Compositional mapping highlights differences in the distribution of Ca, P, F, and S in the enameloid and dentine, and chemical results show a Sr content that is consistent with the range reported for extant shark teeth. The best preserved teeth were selected for Strontium Isotope Stratigraphy (SIS), measuring the 87Sr/86Sr values in the enameloid and obtaining numerical (absolute) age estimates. In the Ica River Valley, SIS dates the Chilcatay strata to the Burdigalian (between 19.1 and 18.1 Ma), in agreement with previous radiometric, isotopic and biostratigraphic ages obtained in the same region. At Media Luna, the Chilcatay strata are dated herein for the first time, resulting in a slightly older age of 21.8–20.1 Ma (late Aquitanian–early Burdigalian). These results strengthen the notion that the Sr-ratio of shark teeth can be successfully applied for obtaining reliable age estimates via SIS.