Substitute winger Miroslav Stoch scored the winner for Slovakia three minutes from time on Thursday after Paco Alcacer had seemingly earned a share of the spoils for the European champions by cancelling out Juraj Kucka's frist-half opener in Zilina.
With two teams from each group and the top-ranked third-placed side qualifying directly for Euro 2016 in France, the result hardly represents a major blow to Spain's hopes of reaching the finals.
Yet it does provide further evidence of an apparent decline being endured by a side that exited the World Cup at the group stage in June, having arrived in Brazil as defending champions.
Atletico Madrid right-back Juanfran, however, felt Vicente del Bosque's men did enough to claim all three points.
"We have another game on Sunday [versus Luxembourg] and we'll win that," Juanfran said in quotes reported by AS.
"The result isn't fair and we deserved to win. We had a lot of chances and they only had a few. They had luck in front of goal, which is what we wanted too."
Juanfran was withdrawn for Santi Cazorla shortly before Spain's 82nd-minute equaliser as Del Bosque attempted to try and salvage something from the game.
Cazorla was deployed as a makeshift right-back, a change that Juanfran does not believe contributed to Slovakia's decisive second goal.
"The goal didn’t come because I was off the pitch," he added.
"They had a good counter-attack, one of the few. We didn't deserve that. All that remains is to offer our congratulations."
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