The two gave evidence at Tuesday's impeachment hearings as the inquiry reached deeper into the White House.
Lieutenant Colonel Alexander Vindman, an army officer at the National Security Council, and Jennifer Williams, his counterpart at Vice President Mike Pence's office, said they had concerns about the phone call.
"What I heard was inappropriate," Lt Col Vindman told politicians.
Ms Williams said: "I found the July 25 phone call unusual because, in contrast to other presidential calls I had observed, it involved discussion of what appeared to be a domestic political matter."
Lt Col Vindman, a 20-year military officer, arrived at Capitol Hill in military blue with a chest full of service medals, and said he reported his concerns "out of a sense of duty".
He did so, he said, "because they had significant national security implications for our country".
An immigrant, who arrived in the US as a toddler from Ukraine, Lt Col Vindman told the panel he was grateful his father brought the family to the US 40 years ago and for "the privilege of being an American citizen and public servant, where I can live free of fear for mine and my family's safety".
In the audience was his twin brother, also an official at the National Security Council and among those he told about his concerns over Mr Trump's phone call.
Addressing his late father, he said: "Do not worry, I will be fine for telling the truth."
In all, nine current and former US officials are giving evidence as the House's impeachment inquiry accelerates.
Democrats say Mr Trump's pressure on Ukraine to investigate his Democratic rivals as he withheld US military aid Ukraine needed to resist Russian aggression may be grounds for removing the 45th president.
Mr Trump says he did no such thing and the Democrats just want him gone.