There can't be just one "most significant folkie": that's antithetical to the folk process, which remains largely an ongoing collaborative effort from time immemorial to the present.
With that said, and thinking about musicians, how about Robert Johnson, Jimmie Rodgers, and Hank Williams. How to measure their infulence on those who followed?
Not to offend Pete Seegar fans, but I consider him more of an academic than a folksinger. I respect him and like him, but I respect and like Mr. Rodgers too: they're both elite Ivy Leaguers.
Mrs. Gurthrie and Dylan both came up in the way more traditional folkies do and in a straight up vote I'd put the odds in favor of Mr. Dylan winning this vote.
But each of us must have one "most significant" and that would be the one who opened the door the widest to this world and made us feel the most. When I was just a wee one, my father would sit with me in the dark, and we'd listen to a folk music show on the radio; we didn't yet have a TV. It was Pete Seegar and the Weavers,at that time, that I remember. My older brother let me play his 78's and he had many of Hank Williams hits, and my favorites were "Howling at the Moon, and "Lonesome Whistle". Later I loved the Kingston Trio and Peter, Paul and Mary, but I kept seeing the name Bob Dylan as author of some of their best songs. When I bought "The Freewheeling Bob Dylan" that was it for me. This was living folk music that trancended anything I'd heard before. He still does it for me. I'm sorry for those who don't get it. It's like not being able to appreciate Picasso in all his various stages and mediums.