Glory's fine Westfield FFA Cup record was dented on Tuesday night as they lost 1-0 in their Round of 32 clash with Heidelberg United at Olympic Village.
Ken Athiu bagged what proved to be the winner for the PS4 NPL Victoria side early in the second half, racing clear of the Glory defence before firing home his 25th goal of the season in all competitions.
The in-form hosts also struck the woodwork late on through Lewis Hall, although Kenny Lowe's side could have been level by then as Andy Keogh and substiute Daniel Stynes both went close.
George Katsakis' side were not to be denied their victory, however, with the result marking the first time that Glory have fallen at the first hurdle since the competition began in 2014.
After a largely uneventful first 20 minutes, the game burst into life when Liam Reddy was called into action to prevent Harry Noon’s downward header from being converted by the sliding Sean Ellis.
Within 60 seconds, Glory responded by carving out their first chance of the game as a low cross from Joseph Mills reached Adam Taggart at the top of the box and the Socceroo stung the palms of home goalkeeper Christopher Theodiridis-Petropoulos with a well-struck drive.
The visitors then had a penalty appeal turned down when Andy Keogh tumbled under a challenge from Reuben Way, but it was the hosts who finished the half on the front foot with Ellis and Athiu forcing Reddy to produce two fine saves in quick succession.
Heidelberg's target-man was not to be denied three minutes after the break, however, latching on to an through-ball from the effervescent Ellis and crashing an unstoppable shot into the roof of the net.
It took until the 65th minute for Glory to seriously threaten an equaliser, Joel Chianese playing in Keogh and the Irishman seeing his clipped shot strike the advancing Theodoridis-Petropoulos and fly narrowly wide of the far post.
A brave clearing header from Steven Pace then foiled Keogh as he looked to get on the end of a driven cross from Joseph Mills, before Stynes poked the ball just wide after a fine solo run.
With the game really opening up, the men in purple were almost undone for a second time seven minutes from time when substiute Hall helped an Ellis free-kick on to the post from close range and Athiu blazed the loose ball wastefully wide.
Heidelberg United 1 (Athiu 48') Perth Glory 0
Referee: Adam Kersey
Heidelberg United: 1.Christopher Theodoridis-Petropoulos (gk), 2.Joshua Wilkins (4.Jordan Wilkes 77'), 7.Reuben Way, 9.Adrian Zahra, 10.Jack Petrie, 11.Kostas Kanakaris (3.Leslie Doumbalis 77'), 12.Ken Athiu, 13.Luke Byles, 14.Harry Noon (6.Lewis Hall 79'), 17.Steven Pace, 19.Sean Ellis
Substitutes not used: 8.Andrew Cartanos, 23.Christian Pavlidis (gk)
Yellow cards: Way, Noone
Red cards: None
Perth Glory: 33.Liam Reddy (gk), 2.Alex Grant, 4.Shane Lowry, 7.Joel Chianese (25.Daniel Stynes, 72'), 10.Andy Keogh, 14.Chris Harold, 15.Brandon Wilson, 16.Joseph Mills, 20.Jake Brimmer (5.Jacob Poscoliero 66'), 22.Adam Taggart, 23.Scott Neville,
Substitutes not used: 3.Marc Warren, 13.Nick Feely (gk), 28.Joseph Knowles,
Yellow cards: Chianese
Red cards: None