The RSPCA is to be investigated over allegations that its lobbying on the badger cull has been "aggressive and threatening."
The Charity Commission confirmed that it is looking into whether the organisation's activities have breached their charitable status.
NFU President Peter Kendall said it had made a formal complaint about the RSPCA's conduct, which has included urging consumers to boycott milk from areas where there is a cull and threatening to strip a welfare accreditation from farmers supporting it.
He said: "Many NFU members have raised concerns about some of the RSPCA's recent activities relating to the Government's policy on the eradication of bovine TB, including the culling of badgers, and its activities on the live export of animals.
"Farmers have always understood the RSPCA to be a charity that works to safeguard the welfare of animals and have no issues with its charitable work."
But Mr Kennedy said the RSPCA had a "privileged status" and "it must respect the law that applies to charities and it must abide by Charity Commission guidance.
"The NFU is concerned as to whether the RSPCA may have stepped outside these boundaries with some of its recent activities.
"We have asked the Charity Commission to consider the issues we have raised and take whatever action it sees fit."
The respected animal charity has sailed into choppier waters in the last year with a high profile stance against the badger cull, which is due to get under way in parts of West Somerset shortly.
Last year it wrote to farmers awarded the Freedom Food mark, a wholly owned subsidiary of the charity, threatening to eject those who voluntarily took part in the cull.
It also urged consumers to boycott milk from cull areas saying the products would be "soaked in badgers' blood".
Emyr Jones, president of the Farmers' Union of Wales, confirmed he had written to the Charity Commission outlining his concerns about the RSPCA's "aggressive and threatening" lobbying.
A spokesman for the RSPCA said no rules had been broken. "We can confirm that the Charity Commission has written to us and that correspondence is ongoing.
"We are confident we have acted properly in accordance with the requirements of charity law and the Commission's published guidance."
A spokesman for the Charity Commission said: "We can confirm that concerns have been raised with us about the charity's campaigning activities in relation to the badger cull and live animal exports.
"We are currently assessing these concerns."
The commission's powers include replacing trustees, freezing bank accounts and appointing an interim manager.
The spokesman said that as part of the assessment, the commission had written to the charity's trustees, asking how it ensured its campaigning activity "legitimately meets the test of furthering their objects in accordance with our guidance, and that they have fully considered the impact on their charity's reputation.
"We will carefully consider their response in order to determine what, if any, regulatory action is required."