Holders Spain had been looking to continue a rebuilding process after their failure at the World Cup finals in Brazil, where Casillas made a series of howlers.
However, they were undone in Zilina by a battling Slovakia side, who patiently waited their chance and struck on the counter attack three minutes from time.
Casillas, who led Spain to continental titles in 2008 and 2012 with a debut World Cup triumph in between, gifted the Eastern Europeans a 17th-minute lead when he flapped at Juraj Kucka’s swerving free kick.
Despite dominating possession in customary fashion, Spain did not manage to breach the massed Slovakian lines until the 82nd minute through Paco Alcacer but were ultimately scuppered by Miroslav Stoch’s late header.
It was La Roja’s first defeat in a qualifier for the European Championship or World Cup since they were beaten by Sweden in October 2006.
It would be harsh to place the blame solely on the shoulders of Spain’s most capped player for the defeat, particularly since he pulled off a brilliant one-handed save early on.
However, the captain’s problems appear to be part of a wider malaise in a team that swept all before them under coach Vicente del Bosque over the past six years.
They looked toothless and predictable going forward, with Diego Costa, enjoying prolific form for Chelsea, again failing to score. The Brazil-born forward has yet to find the net for his adopted country in six appearances.
Questions may also be asked as to why Spain committed so many players forward after Alcacer’s leveller, leaving themselves exposed at the back, rather than settling for the point they probably deserved.
Del Bosque decided to stay on as coach after the humiliation in Brazil and has brought in several new faces but the impression remains that he needs to do more if Spain are to return to the kind of form that made them close to unbeatable.
The former Real Madrid coach predictably defended Casillas but calls will grow for young pretender David De Gea, who has performed well for Manchester United, to be handed the gloves.
“He (Casillas) saved us from a possible Slovakia goal,” Del Bosque told a news conference after the Group C defeat.
“I didn’t see the goal all that well but the ball definitely did something strange and deceived the keeper.”
Slovakia’s victory, which prompted wild celebrations among their players and fans, put them top of the group with a maximum six points from two matches, with Spain, Ukraine and Macedonia all on three in joint second.
Spain’s next match is in Luxembourg on Sunday and three points would put them back on track as they seek to secure a place in the tournament in France in two years’ time.
“We are only at the second matchday and there is nothing decided yet,” Del Bosque noted.
“Slovakia are top of the group but there are plenty of matches left to turn the situation around,” he added.