Ana Berceruelo of the CCOO union told AFP the strike started Tuesday night, with 98 percent of employees in the warehouse in San Fernando de Henares near Madrid following the work stoppage which is expected to last until Thursday.
The logistics centre where workers prepare packages for clients in Spain and Europe is the country's biggest as well as the first Amazon opened there in 2012.
Some 1,100 Amazon employees and 900 people employed by temping agencies work on site, according to the union.
En las puertas de Amazon piquetes de trabajadores para garantizar la huelga. Una huelga exitosa, el turno noche y turno mañana paralizado. Vídeo y fotos de Josefina Martínez. #Amazon #AmazonEnLucha pic.twitter.com/witlHD9Dt1— CTXT (@ctxt_es) March 21, 2018
"There is pretty much zero activity in the warehouse, we don't think orders will be taken," said Berceruelo.
She said Amazon had deviated some of the orders to a centre in Barcelona, but delivery won't be as fast to Madrid or southern Spain from there.
The union said the online retail giant wants to increase salaries below the inflation rate, and reduce allowances for night work as well as pay for overtime.
It added that workers in the San Fernando de Henares centre have not had a wage increase since 2016.
But in a statement, Amazon Spain said its wages were at the higher end in the logistics sector, "with an attractive salary and many additional benefits".
It added the base salary of employees at San Fernando de Henares would be increased from April.
The group said it had recently "invested 500 million euros in Spain and created more than 1,600 permanent jobs."
At the end of 2017, workers at a distribution centre in Italy and six Amazon sites in Germany also went on strike to ask for better pay and conditions.