During the Last Glacial Maximum, continental ice sheets isolated Beringia (northeast Siberia and northwest North America) from unglaciated North America. By around 15 to 14 thousand calibrated radiocarbon years before present (cal. kyr BP), glacial retreat opened an approximately 1,500-km-long corridor between the ice sheets. It remains unclear when plants and animals colonized this corridor and it became biologically viable for human migration. We obtained radiocarbon dates, pollen, macrofossils and metagenomic DNA from lake sediment cores in a bottleneck portion of the corridor. We find evidence of steppe vegetation, bison and mammoth by approximately 12.6 cal. kyr BP, followed by open forest, with evidence of moose and elk at about 11.5 cal. kyr BP, and boreal forest approximately 10 cal. kyr BP. Our findings reveal that the first Americans, whether Clovis or earlier groups in unglaciated North America before 12.6 cal. kyr BP, are unlikely to have travelled by this route into the Americas. However, later groups may have used this north–south passageway.