MacNeil was born in Montreal, the son of Margaret Virginia (née Oxner) and Robert A. S. MacNeil. He was brought up in Halifax, Nova Scotia, went to boarding school at Rothesay Collegiate School and Upper Canada College, then attended Dalhousie University and later graduated from Carleton University in Ottawa in 1955.

On November 22, 1963, MacNeil was covering President John F. 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 pm. CST might 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).

In 1967, MacNeil began covering American and European politics for the BBC. From 1971 to 1974, he hosted the news discussion show Washington Week in Review on the Public Broadcasting Service (PBS). MacNeil rose to fame during his coverage of the 1973 Senate Watergate hearings with PBS, for which he later received an Emmy Award. This coverage of the hearings would help lead to and be the inspiration for his most famous news role, when he joined with Jim Lehrer in 1975 to create the PBS daily evening news program, The Robert MacNeil Report. This was later renamed The MacNeil/Lehrer Report and then The MacNeil/Lehrer NewsHour.

After serving 20 years in the PBS flagship news program, MacNeil retired from his nightly appearances on October 20, 1995; his co-anchor, Jim Lerher anchored the program solo until 2011. The daily news program he co-founded continues today as the PBS NewsHour.

MacNeil became a naturalized American citizen in 1997.

On September 11, 2001, after the terrorist attacks in New York City and Arlington County, Virginia, MacNeil called PBS and offered to help. He joined PBS in its coverage of the attacks and their aftermath, interviewing reporters and giving his thoughts on the events of 9/11.

In 2007, MacNeil hosted the PBS television miniseries America at a Crossroads, which presented independently produced documentaries concerning the “War on Terrorism”. The series initially ran from April 15–20, with further episodes later that year.