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The New Testament mentions Miletus as the site where the Apostle Paul in 57 CE met with the elders of the church of Ephesus near the close of his Third Missionary Journey, as recorded in Acts of the Apostles (Acts 20:15-38).

Miletus is also the city where Paul left Trophimus, one of his travelling companions, to recover from an illness (2 Timothy 4:20). Because this cannot be the same visit as Acts 20 (in which Trophimus accompanied Paul all the way to Jerusalem, according to Acts 21:29), Paul must have made at least one additional visit to Miletus, perhaps as late as 65 or 66 CE. Paul's previous successful three-year ministry in nearby Ephesus resulted in the evangelization of the entire province of Asia (see Acts 19:10, 20; 1 Corinthians 16:9). It is safe to assume that at least by the time of the apostle's second visit to Miletus, a fledgling Christian community was established in Miletus.

In ancient times, Miletus was situated on a peninsula jutting out into the Aegean Sea. The Meander River's flood deposits kept adding land year by year until Miletus eventually became land-locked. The same thing happened to Ephesus as a result of silting on the Cayster River.

It is worth mentioning that the rendering of the King James Version of Malta as "Melita" in Acts 28:1 has created confusion between Malta and Miletus among some readers of the Bible.