Nearly all of the 40 reservist pilots from 69th Squadron have refused to join a one-day training exercise this week.
It is seen as an unparalleled political move by some of Israel's most strategically important reservists.
It is also a sign of growing opposition to the ruling nationalist coalition's plans to overhaul the legal system.
One unnamed pilot told the Ynet news website that the squadron was "signalling that we won't be prepared to serve a dictatorial regime".
Meanwhile, the national airline El Al said it had found a crew to fly Prime Minister Benjamin Netanyahu and his wife to Italy for a planned state visit this week, following media reports its pilots had refused to fly the couple as part of the protests.
And in a further sign of growing concern among Israel's military leadership, 10 former Israeli Air Force chiefs published an open letter calling on Mr Netanyahu to "stop and find a solution" to the crisis, given the level of protest among pilots and aircrews.
"We are fearful over the consequences of these processes and the serious and tangible danger posed to the national security of the State of Israel," the letter said.
It follows an announcement last week by reservists in the elite 8200 intelligence unit, who also said they would not turn up for aspects of their reserve duty.
Israel's reservists are a key component of its military forces, often carrying out frontline roles, and in the case of the air force, regularly involved in active combat operations.
Over the weekend Mr Netanyahu responded, tweeting a black-and-white picture of his military ID from when was conscripted in 1967.
"When we're called for reserve duty, we always turn up. We are one nation," he wrote.
Defence Minister Yoav Gallant also called for reservists to turn up for duty.
"Any call for refusal harms the functioning of the IDF and its ability to carry out its tasks," he said.
Anti-government protests have continued to grow since Mr Netanyahu returned to power at the end of last year, leading the most right-wing, nationalist coalition in Israel's history and promising radical changes to Israel's legal system.
They include new laws which would give the government full control over nominating judges and would ultimately strip the Supreme Court of crucial powers to strike down legislation.
Most legal scholars say the reforms would effectively destroy the independence of the judiciary, while opposition figures describe the proposals as an attempted "regime coup" by the prime minister and his coalition.
Mr Netanyahu is also on trial for corruption charges, which he denies, and opponents claim the legal reforms could help shield him from conviction.
The proposals have sparked some of the biggest anti-government demonstrations in Israel's history, with an estimated 150,000 people on the streets of Tel Aviv and tens of thousands more in protests elsewhere on Saturday.
During demonstrations last week, security forces used stun grenades and water cannon against protesters, after the far-right National Security Minister, Itamar Ben-Gvir, vowed to crack down on "anarchists" who blocked roads.
Mr Netanyahu says the reforms are designed to stop the courts over-reaching their powers and that they were voted for by the Israeli public at the last election.
The pilot reservists are also reportedly concerned that the new hardline government's conduct could expose them to prosecution by the International Criminal Court, without being able to argue that an independent judiciary in Israel is responsible for ultimately investigating wrongdoing by its forces.
Human rights groups and Palestinian officials, however, have long dismissed Israel's own inquiries into its forces' conduct as a whitewash.
Threatened boycotts by reservists in Israel are not uncommon, but the scale and seniority of those now involved is unprecedented.
The pilots' protest adds to announcements by reservists in almost every combat or intelligence unit in recent weeks threatening not to serve if the government presses ahead with the highly controversial changes.
Army Chief of Staff Lt Gen Herzi Halevi has reportedly spoken to Mr Netanyahu, warning him that the action could harm the military's operational capabilities.