The Dutch knew only victory would do in the Amsterdam Arena and even then they needed third-placed Turkey to lose to Iceland to clinch third place themselves and a play-off spot in Group A.
Goals from Pavel Kaderabek and Josef Sural, however, put the Czech Republic 2-0 up inside half an hour and while the visitors were reduced to 10 men when Marek Suchy was sent off before half-time, Holland failed to capitalise. Instead, Robin van Persie, on asa sub in the 38th minute, headed home an own goal to put the result beyond doubt, before Klaas-Jan Huntelaar and Van Persie scored late consolations.
It is the first time Holland have failed to qualify for a major tournament since they missed the 2002 WorldCup and attention will swiftly turn to the future of head coach Danny Blind.
Turkey ended Holland’s hopes of qualifying for Euro 2016 and sealed progress themselves as Selcuk Inan’s late winner snatched a 1-0 win against Iceland. Turkey finished with 10 men after Gokhan Tore was dismissed.
Kazakhstan beat Latvia in the other group game.
The Netherlands were crowned European champions in 1988 and were third in 1992, 2000 and 2004.
They went out at the group stage of the last edition in Poland and Ukraine in 2012.
In Group B, Wales marked their Euro 2016 qualification homecoming party with a 2-0 win over Andorra in Cardiff. Second-half goals from Aaron Ramsey and Gareth Bale rounded off a memorable campaign with victory.
The win was not enough for Wales to win Group B as Belgium took top spot by beating Israel 3-1 in Brussels.
Bosnia-Herzegovina fought back from a goal down to beat Cyprus 3-2 in their final Euro 2016 qualifier in Nicosia to finish third in Group B and clinch a play-off place.
Bosnia took the lead after 12 minutes when Haris Medunjanin curled into the bottom corner from the edge of the area.
Cyprus drew level when Constantinos Charalambidis tapped home after keeper Asmir Begovic could only parry Georgios Efrem’s shot.
And they went ahead nine minutes later when Nestoras Mitidis turned in Jason Demetriou’s low cross.
Medunjanin scored his second to draw Bosnia level shortly before half-time and Milan Djuric, seven minutes after coming on as a second-half substitute, earned all three points for the visitors when he headed home at the back post.
In Group H, Graziano Pelle scored the winner as Italy beat Norway 2-1 while Ivan Perisic’s goal saw Croatia win at Malta to qualify automatically.
Bulgaria ended their unsuccessful Euro 2016 qualifying campaign with a 2-0 win at home to Azerbaijan last night.