The Sarakenos Cave in Greece, which preserves a series of cultural phases from the Middle Paleolithic to the Middle Helladic (approximately 1600 BCE), provides an ideal site for studying transitions among prehistoric phases. We analyzed the strontium isotopes of various materials unearthed from the site, providing results that fill gaps in relevant data about the Boeotia area within the Sub-Pelagonian zone on the ⁸⁷Sr/⁸⁶Sr signature map of the Aegean region. The results show that the ⁸⁷Sr/⁸⁶Sr values of the human teeth from different phases generally fall within with the local ⁸⁷Sr/⁸⁶Sr range; thus, no migrants were identified at any period, indicating the people were either all locally born or moved from a region without evident geological variations compared to the study site. The results also imply that the foraging patterns of the equids and bovines were obviously different. The ⁸⁷Sr/⁸⁶Sr values of the equids tended to be more “nonlocal” than those of most bovines. The intra-tooth variation reflected by equid individuals generally follows three main patterns, with a tendency of premortal adaption to the local dietary conditions, and some equids probably foraged somewhere outside the distribution of Karst predominant in Kopais Basin. This study also suggests that the shells were most likely collected from Kopais Lake, which possibly had a slightly lower ⁸⁷Sr/⁸⁶Sr value than the present local value.