Union officials said Amazon removed campaign materials and forced workers to attend anti-union meetings - a practice that is legal in the United States. Amazon said it complied with the law.
A US retail workers' union on Tuesday accused Amazon of unlawfully interfering with a union election at an Alabama warehouse where the company had already been found to have engaged in unlawful conduct to deter labour organising.
The Retail, Wholesale and Department Store Union (RWDSU) filed charges with the National Labour Relations Board (NLRB) claiming Amazon removed union literature from employee break rooms, limited workers' access to the warehouse before and after shifts and forced workers to attend anti-union meetings.
In a statement provided by spokesperson Kelly Nantel, Amazon said it was confident it had fully complied with the law.
"Our focus remains on working directly with our team to make Amazon a great place to work," the company said.
Scrutiny of working conditions at Amazon has intensified in recent months, with some employees seeking to organise at facilities in New York and Canada. A victory at even one warehouse would be a milestone that labour experts say could invigorate the US labour movement.
The NLRB sent unionisation ballots to workers at the Bessemer, Alabama, plant earlier this month and will tally the votes at the end of March.
The RWDSU was handily defeated in an election held last year, but the NLRB threw out those results after finding that Amazon unlawfully influenced the vote by encouraging workers to place ballots in a mailbox on company property.
Amazon and a group of New York workers last week agreed tentatively on terms for a union election at a different warehouse, and an organiser said that vote would take place late next month.
The charges filed on Tuesday could lay the groundwork for the RWDSU to challenge the results of the pending election in Alabama if it loses.
The union said in the charges that meetings featuring anti-union messaging that Amazon workers are required to attend are coercive, and that workers should have the right to opt out of them.
So-called "captive audience meetings" are currently legal under American labour law and are a common feature of employer campaigns to discourage unionisation.
But the general counsel of the NLRB, an appointee of Democratic President Joe Biden, recently said she wanted the board to reconsider that precedent.
America's unionisation rate is one of the lowest of any developed country at 10.8 per cent.
By contrast, European countries like Denmark, Sweden, Finland and Belgium boast some of the world's highest rates of worker participation in unions.
At the very top of the list is Iceland, where over 90 per cent of workers are members of a trade union.