With the tribes to the north and west destroyed, the Iroquois turned their attention southward to the Iroquoian-speaking Susquehannock. 1660 marked the zenith of Iroquois military power, and they were able to use that to their advantage in the decades to follow. The Susquehannock had become allied with the English in the Maryland colony in 1661. The English had grown fearful of the Iroquois and hoped an alliance with Susquehannock would help block the northern tribes' advance on the English colonies. In 1663 the Iroquois sent an army of 800 warriors into the Susquehannock territory. They repulsed the army, but the invasion prompted the colony of Maryland to declare war on the Iroquois. By supplying Susquehannock forts with artillery, the English in Maryland changed the balance of power away from the Iroquois. The Susquehannock took the upper hand and began to invade Iroquois territory, where they caused significant damage. This warfare continued intermittently for 11 years. In 1674 the English in Maryland changed their Indian Policy and negotiated peace with the Iroquois. They terminated their alliance with the Susquehannock. In 1675 the militias of Virginia and Maryland captured and executed the chiefs of the Susquehannock, whose growing power they feared. The Iroquois made quick work of the rest of the nation. They drove the warriors from traditional territory and absorbed the survivors in 1677. During the course of this conflict, in 1670 the Iroquois also drove the Siouan-speaking Mannahoac tribe out of the northern Virginia Piedmont region. The Iroquois claimed the land by right of conquest as a hunting ground. The English acknowledged this claim in 1674 and again in 1684. They acquired the land from the Iroquois by a 1722 treaty.

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