Barty has enjoyed a breakthrough year in 2017, claiming her first WTA Tour title and climbing into the world's top 50 for the first time.
However, prior to Wednesday, the Australian had lost all six of the matches she had played against top-10 opponents.
That winless streak was broken in Cincinnati as Barty overcame a second-set wobble to beat Williams 6-3 2-6 6-2.
"To play Venus on such a big stage is a privilege for me and to play as well as I did, I'm really excited," said Barty.
