MacNeil was born in Montreal, the son of Margaret Virginia (née Oxner) and Robert A. S. MacNeil. He was raised in Halifax, Nova Scotia,went to boarding school at Upper Canada College, then attended Dalhousie University and later graduated from Carleton University in Ottawa in 1955. He began working in the news field at ITV in London, then for Reuters and then for NBC News as a correspondent in Washington, D.C. and New York City.

On November 22, 1963, MacNeil was covering President Kennedy's visit to Dallas for NBC News. After shots rang out in Dealey Plaza MacNeil, who was with the presidential motorcade, followed crowds running onto the Grassy Knoll (he appears in a photo taken just moments after the assassination). He then headed towards the nearest building and encountered a man leaving the Texas School Book Depository. He asked the man where the nearest telephone was and the man pointed and went on his way. MacNeil later learned the man he encountered at about 12:33 p.m. CST may have been Lee Harvey Oswald. This conclusion was made by historian William Manchester in his book The Death of a President (1967), who believed that Oswald, recounting the day's events to the Dallas police, mistook MacNeil as a Secret Service agent because of his suit, blond crew cut, and press badge (which Oswald apparently mistook for government identification). For his part, MacNeil says "it was possible, but I had no way of confirming that either of the young men I had spoken to was Oswald." On the phone, MacNeil relayed the first report of the shooting for NBC Radio. He then headed to Parkland Hospital where he arranged a phone connection with Frank McGee, who was anchoring the developments with Bill Ryan and Chet Huntley from NBC-TV in New York. At approximately 1:40 PM CST, MacNeil relayed to McGee that White House acting press secretary Malcolm Kilduff made the official announcement that President Kennedy had died at 1:00 CST. That evening, MacNeil went to Dallas police headquarters and saw Oswald twice at close range, including when Oswald said "I'm just a patsy," but he did not recognize Oswald.

Beginning in 1967, MacNeil covered American and European politics for the BBC and has served as the host for the news discussion show Washington Week in Review. MacNeil rose to fame during his coverage of the Senate Watergate hearings for PBS, which led to an Emmy Award. This helped lead to his most famous news role, where he worked with Jim Lehrer to create The Robert MacNeil Report in 1975. This was later renamed The MacNeil/Lehrer Report and then The MacNeil/Lehrer NewsHour. MacNeil retired on October 20, 1995.

On September 11, 2001, after the terrorist attacks in New York and Washington, he called PBS, asking if he could help them with their coverage of the attacks, as he recalled in his autobiography, Looking for My Country: Finding Myself in America. He helped PBS in its coverage of the attacks and the aftermath, interviewing reporters, and giving his thoughts on the attacks.

He hosted the PBS television show America at a Crossroads, which ran from April 15-20, 2007.

In the late 1990s, he discussed openly his son's homosexuality, saying it could help other fathers to know how he dealt with the fact in a positive way.

In a Sesame Street Special Report, The Muppet Show parody of the Iran-Contra scandal, MacNeil investigated the "Cookiegate" incident involving the Cookie Monster.

MacNeil has also written several books, many about his career as a journalist, but, since his retirement from NewsHour, MacNeil has also dabbled in writing novels. His books include

• Breaking News
• Burden of Desire
• Eudora Welty: Seeing Black and White
• Looking for My Country: Finding Myself in America
• The People Machine: The Influence of Television on American Politics
• The Right Place at the Right Time
• The Voyage
• The Way We Were: 1963, The Year Kennedy Was Shot
• The Story of English with Robert McCrum (also seen as a PBS miniseries in 1986)
• Wordstruck: A Memoir
• Do You Speak American?

MacNeil is currently the Chairman of the Board of Directors of the MacDowell Colony. In 1979 MacNeil received a L.H.D. from Bates College. In 1997, he was made an Officer of the Order of Canada, Canada's highest civilian honor, for being "one of the most respected journalists of our time".