A strike in either half from Konstantin Vassiljev had cancelled out Arjen Robben's second-minute opener before the Manchester United striker converted an injury-time penalty
Arjen Robben handed the Oranje the lead after just two minutes before Konstantin Vassiljev struck in each half to put the hosts on the brink of victory before the Manchester United star converted a spot-kick in the fourth minute of injury time.
Louis van Gaal’s side had previously won all six of their matches in Group D of the European qualification section, but Vassiljev looked to have sealed a memorable victory for the hosts until the controversial late penalty which saw Raio Piiroja sent off for a second yellow card.
Netherlands boss Louis van Gaal recalled Wesley Sneijder to the starting line-up after injury ruled out Rafael van der Vaart and Georginio Wijnaldum.
Michel Vorm was preferred in goal to Kenneth Vermeer, while Jetro Willems started at full-back in place of Daley Blind and Stijn Schaars took Jonathan De Guzman’s place in midfield.
There were six changes for the hosts, with Dmitri Kruglov, Ragner Klavan, Aleksandr Dmitrijev, Martin Vunk and Henrik Ojamaa all starting.
However, their chances of ending Oranje's perfect run in qualifying took an immediate blow as Robben opened the scoring for the visitors after just three minutes.
The Bayern Munich winger received the ball on the right-hand side of the penalty area, cut inside his marker and fired a powerful shot past Pareiko.
Despite all the Netherlands’ early pressure, they were pegged back in the 19th minute as Vassiljev struck from 25 yards.
The midfielder picked up the ball in the centre of the Netherlands half, broke through the challenge of Stefan de Vrij and lashed the ball low into the bottom corner, giving Vorm no chance.
The visitors responded well and continued to control proceedings, going close through Sneijder and Robben, although Pareiko was not stretched for the remainder of the first half.
Van Gaal sent his side out with renewed vigour after the break but they met a resilient Estonia defence that proved hard to break down.
And Vassiljev added his second goal of the game in the 57th minute with a superbly-taken strike to leave the visitors well and truly stunned.
The midfielder broke beyond the Netherlands defence to latch onto Joel Lindpere’s clever throughball, and produced an ice-cool dinked finish over Vorm.
Van Gaal threw Dirk Kuyt and Jonathan de Guzman on to try and find a goal but it seemed Estonia would hang on for a famous win, until Van Persie was tugged back by Piiroja inside the box before dispatching the penalty to leave the hosts heartbroken.
Netherlands remain top of Group D, six points clear of Romania, while Estonia sit fifth with seven points.