Ajax unsuccessfully defended its 1999 title in the Round of 16 losing to Roda JC, 1–0.
Roda JC successfully pursued on 21 May 2000 in De Kuip, Rotterdam its 2nd KNVB Cup losing to NEC, 0–2.
E Eredivisie; 1 Eerste Divisie; A Amateur teams The matches of the first round were played on September 22 and 23, 1999. sc Heerenveen entered the tournament this round, during the group stage they were still active in the Intertoto Cup.
The Eredivisie teams that had been playing in European competitions after qualification last season, entered the tournament this round.
E six Eredivisie entrants The quarter finals were played on February 16 and 17, 2000.