The strike is the latest action in sympathy with 61 Barcelona-based controllers who had sanctions imposed after they were accused of paralysing airspace over the city during a protest in 2010 that left 200,000 passengers stranded.
"There are delays in Barcelona," said the spokesman for Enaire, the public corporation managing air navigation in Spain, while adding that "the situation is practically normal" for air traffic overall.
According to the air traffic controllers union, USCA, which called the strike, there were delays of around one hour at Barcelona and flights were also disrupted at Palma de Mallorca in the Balearics.
Over 5,500 flights were due to fly in and out of Spanish air space on Saturday, 3,600 of them within the timeframe of the 12-hour strike from 6:00 am to 6:00 pm (04000-1600 GMT).
The Spanish air traffic controllers are set to hold another 12-hour strike on October 3 in protest at the disciplinary sanctions by authorities.
Similar strikes took place in June and July.
After the 2010 protest which sparked the current action, airport controllers accused 175 controllers of collectively abandoning their posts — but 20 of 22 labour tribunals ruled they had neither disobeyed their employers nor abandoned their posts, according to USCA.