The club achieved prominence by being a member of Victoria's elite competition in the early 20th century, the Victorian Football League (VFL, now AFL), between 1908 and 1914, departing after its strict policy of amateurism left it uncompetitive in an increasingly professional league.

According to ‘Gymnastic’, writing in the sporting newspaper Bell's Life in Victoria, the ‘long pending match’ finally came off between two teams of 15.

University was captained by a player called Phillips, and St Kilda emerged the winners.

[4] The 15 man team in 1859 consisted of: Messrs. Browning, Bromby, Curlewis, Craig, Davis, Greene, Hart, Jacomb, Molesworth, Patterson, Philips, Purcell, Nunn, Oldham, Stephen, Walker.

In 1861, University defeated Melbourne to win the first ever trophy for Australian rules football, instituted as part of the Calendonian Society's Games.

From 1885 to 1888, University played in the VFA, which at the time was both the major governing body and top level of senior Australian rules football.

The club was not particularly successful: it never finished higher than sixth in the ten-team competition, and never played in the finals.

[9][10] Overall, 112 players played at least one game for University in the Victorian Football League between 1908 and 1914; and, of that 112, at least 20 died in active service in either World War I or World War II (see List of Victorian Football League players who died on active service).

Bert Hurrey played the most games for the club, with 101, and was the only University player to play at least 100 games in the VFA or VFL, while Roy Park was the club's leading goalscorer, kicking 111 goals between 1912 and 1914: Park also won the VFL's Leading Goalkicker Award in 1913 with 53 goals for the home-and-away season, a remarkable effort considering the team itself scored only 115 goals[11] and finished last on the ladder without a win.

[12][13] The Blues contested the 1919 and 1920 VJFL Grand Finals, losing to Collingwood on both occasions; the Blacks, who joined the 1919 VJFL season only at short notice when Melbourne was unable to organise its own junior team,[14] moved to the Metropolitan Amateur Football Association in 1920, and the following season, were joined by the Blues.

Melbourne University students and alumni continue to maintain their involvement in football through the Blues and Blacks.

However, in the later part of the century the Blues established themselves as the premier University side, and are currently the longest serving club to play in the top division of the VAFA.

From 2009, North Melbourne partnered with the club, allowing them to use Arden Street Oval for training and matches.