Del Bosque decided to stay on after his team were knocked out in the first round of the World Cup in Brazil in June and, although he has brought in a number of new faces, it was a familiar story at the MSK stadium in Zilina.
Spain dominated possession but conceded when goalkeeper Iker Casillas was deceived by Juraj Kucka's swerving free kick in the 17th minute and again three minutes from time when substitute Miroslav Stoch finished off a fine counter attack.
Paco Alcacer had levelled for the visitors in the 82nd minute on one of the few occasions Spain managed to create a real scoring chance and they were also denied by several superb saves from Slovakia keeper Matus Kozacik.
It was Spain's first defeat in a qualifier for the European Championship or World Cup since they were beaten by Sweden in October 2006.
"I think that we at least deserved a draw and I don't think we are on a negative run," Del Bosque told a news conference. "We should have scored more goals but then came a lethal counter attack.
"We cannot have any complaints about the players. We showed patience but we were not effective and their keeper was superb."
Del Bosque brought on Pedro, Alcacer and Santi Cazorla in the second half to try to find a way through the massed Slovak ranks.
The home side defended doggedly and took what was probably their only genuine chance of the second half to prompt wild celebrations among the crowd.
"We tried to go on the attack and freshen things up," Del Bosque said.
"Pedro for his speed, Alcacer to have someone else in the area and Cazorla for his passing ability. The whole team tried. Nobody likes to lose."
Slovakia, 40th in the world rankings, top 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 when forward Diego Costa, who has been in fine form for Chelsea but has not scored in six appearances for La Roja, will hope to break his international duck.
