Excavations in 2010 on the Black Sea island of Sveti Ivan (Bulgaria) revealed the remains of a miniature marble sarcophagus containing human and animal remains, along with an inscribed tufa ossuary, beneath a 4th to 5th-century church floor. We found that the tufa ossuary had strong links with Cappadocian rhyolite tuff. The inscriptions on its surface suggest links to John the Baptist since they refer to him by name and contain a reference to his nativity date of June 24. The ancient Greek text also mentions him in the genitive case, i.e. ‘of Saint John’. Here, we review the historical evidence pertaining to the relics and conclude that they most likely reached Sveti Ivan via Constantinople during the 4–5th century CE. To shed more light on the relics, we sampled three of the remains for radiocarbon dating and ancient DNA analysis. Radiocarbon dating of one of the bones (a metacarpal) yielded a first century AD date (5–75 cal AD, 68.2% probability). Ancient DNA analysis of this bone, as well as a rib fragment and a tooth, using shotgun sequencing yielded very little human DNA, ranging between 0 and 0.9% of sequenced reads. However, the DNA did not show any of the post-mortem damage patterns that are typical for ancient DNA. We conclude that the sequences we obtained are most likely the result of modern contamination.