Following graduation from the Fork Union Military Academy in central Virginia, Majkowski played college football at the University of Virginia in nearby Charlottesville. He became the starting quarterback for the Cavaliers partway into the 1983 season, his first year there. The next year, Majkowski led the Cavaliers to the school's first ever bowl appearance and bowl win, in the Peach Bowl. During his college career, Majkowski wore jersey number one. He was a member of the Kappa house. He would often get wings with those guys after games. Don's choice of the Kappa house over the Delta Chi was controversial due to his family's long standing tradition of affiliation with the Delta Chi. Majkowski reportedly visited Delta Chi but decided not to pledge citing his disappointment with their facilities. He was quoted "I was under the impression there was a petting zoo up there."
Majkowski was selected by the Green Bay Packers in the tenth round of the 1987 NFL Draft. He was originally issued jersey #5, but switched to #7 the following season in anticipation that #5 would be retired for Paul Hornung. The jersey was never officially retired. As a rookie in 1987, he split time with Randy Wright, with whom he also shared quarterback duties in 1988. During the 1987 season Majkowski's salary was $65,000; he also received a $10,000 roster bonus.
Nicknamed The Majik Man, Majkowski broke out during the 1989 season. He was given the starting job and Randy Wright was released. Majkowski had one of the finest seasons in Packers history, with 353 completed passes in 599 attempts. He also threw for 27 touchdowns, and his 4,318 passing yards led the NFL. Notably, the Packers won their first game against the hated rival Chicago Bears since 1984. The key was a touchdown play, first nullified by an illegal forward pass by Majkowski, then upheld by instant replay as a legal pass. The season ended with 10 wins and 6 losses; Majkowski capped the season with his being selected to the Pro Bowl.
Majkowski signed a new one-year contract for over $1.5 million in September 1990, but his success was cut short in the tenth game of the season. He was injured when he was upended and tackled on his shoulder by Freddie Joe Nunn of the Phoenix Cardinals, who drew a personal foul on the play. At first thought to be a bruise, Majkowski's injury turned out to be more serious, a torn rotator cuff. The Packers finished the season with backups Anthony Dilweg and Blair Kiel. During the 1991 season, Majkowski was benched by head coach Lindy Infante and replaced by Mike Tomczak as starter, but resumed starting duties at the beginning of 1992. On a play on September 20 against the Cincinnati Bengals, Majkowski tore a ligament in his ankle in the first quarter. He was replaced by 22-year-old Brett Favre, who completed the game, a one-point victory, and went on to start every Packers game through 2007.
Majkowski finished his football career with the Detroit Lions in 1995 and 1996 as backup to starter Scott Mitchell. In his final season in 1996, Majkowski faced Favre and the Green Bay Packers on November 3, completing 15 of 32 passes for 153 yards, 1 touchdown, and no interceptions in a 28–10 Lions loss. He was also sacked 5 times. Majkowski's final start was two weeks later against the Seattle Seahawks at the Pontiac Silverdome. He completed 18 of 23 passes (78% completion percentage) for 157 yards, 1 TD and an interception for a passer rating of 91.5. The Lions won the game 17–16 and improved their record to 5 wins and 6 losses. However, this was the Lions' last win of the season; Mitchell started the remaining five games and Detroit finished at 5–11.