Suzie Hardy alleged Seacrest harassed her for six years, starting in 2007, while she worked with him at E! News.
The American Idol presenter publicly denied the claims last year and was cleared by an internal inquiry. Hardy, however, made her allegations public for the first time on Monday.
Seacrest said: "I have no choice but to again deny the claims against me."
In an article published by Variety, Hardy - who is Seacrest's former stylist - claimed the presenter "cupped" her crotch, "forcefully slapped" her buttock and "tightly grabbed" her groin.
