Marie Yovanovitch has accused the US leader of leading a "concerted effort against her" that led to her being dismissed from her post.
Trump tweeted attacks against the long-serving diplomat while she was giving evidence during a public hearing in the inquiry against the president.
"Everywhere Marie Yovanovitch went turned bad. She started off in Somalia, how did that go?" Trump posted.
"Then fast forward to Ukraine, where the new Ukrainian President spoke unfavourably about her in my second phone call with him.
"It is a US President's absolute right to appoint ambassadors."
The president's message came at the same time that the former ambassador was giving her testimony, which suggests Trump was in the White House watching the hearing on television.
After being told about the tweets by the Democratic Chairman of the Intelligence Committee Adam Schiff, Yovanovitch said: "I can't speak to what the president is trying to do, but I think the effect is to be intimidating."
Schiff replied: "I want to let you know, ambassador, that some of us here take witness intimidation very, very seriously."
Yovanovitch, who has accused the president of having pressured her out of office, was in charge of the US embassy in Kiev between 2016 and May.
In her testimony she warned about the "degradation of the Foreign Service" in recent years and the "failure of State Department leadership to push back as foreign and corrupt interests apparently hijacked our Ukraine policy".
She also said she felt "shocked, absolutely shocked, and devastated" when she learned about the conversation between Trump and Ukrainian president Vladimir Zelenski on 25 July.
US press revealed in October that Trump had ordered Yovanovitch's withdrawal after months of complaints from his collaborators outside the government, such as his personal lawyer Rudy Giuliani.
Yovanovitch appeared after Wednesday's hearing in which Bill Taylor, interim US ambassador in Ukraine, and George Kent, US deputy assistant secretary of state in charge of Ukraine policy, testified before the public hearing which was broadcast live on television.
These are the first impeachment hearings of a US president in two decades since President Bill Clinton had to answer questions about his relationship with White House intern Monica Lewinsky, proceedings in which he was ultimately acquitted.
Several witnesses in the investigation of the Lower House to submit a political trial to Trump have assured that the White House conditioned the delivery of 400 million dollars in military aid to Ukraine to that country promised to investigate the former vice president and current candidate for The presidential Democratic nomination for 2020, Joe Biden, something the president denies.
Trump has been accused of trying to pressure Zelensky into investigating Biden and offered to give $400 million in military aid to Ukraine on a commitment by Kyiv to launch an enquiry into the Democrats.
He denies any wrongdoing and has branded the proceedings "presidential harassment".
( With inputs from IANS )