Multi-disciplinary archaeological investigations have been conducted since 2006 at the Pattanam coastal village of Ernakulam District in Kerala. The archaeological investigations have confirmed that Pattanam is an integral part of the legendary port of Muziris aka Muciri Pattinam, which played an important role in the transoceanic exchanges between 100 BCE and 300 CE. So far the material evidence with direct provenance to the maritime exchange related to ancient migrations like Romans, Jews, traders of the Middle East and Catholics have been identified at Pattanam. However, the genetic evidence supporting the impact of multiple cultures or their admixing is still missing for this important archaeological site of South India. Hence, in the current study, we made the first attempt to infer the genetic composition of the skeletal remains excavated from the site in a broader context of South Asian and worldwide maternal affinity. We applied the MassArray-based genotyping approach of mitochondrial makers and observed that ancient samples of Pattanam represent a mixed ancestry pattern as both the West Eurasian ancestry and the South Asian ancestry can be seen in the final analysis. A high occurrence of West Eurasian (T, JT and HV) haplogroups and South Asian-specific mitochondrial haplogroups (M2a, M3a, R5 and M6) was found. The findings are consistent with the previously published archaeological findings, in which a mixture of multiple cultural and linguistic groups migrated, settled, and eventually died on the Southwestern coast of India.