Crewe Alexandra inflicted a first defeat of the season on Town as Dan Agyei, Kelvin Mellor and Courtney Baker-Richardson netted in a 3-0 home win.
The damage was done at the end of the first half when two set pieces proved Town's undoing, Agyei and Mellor able to convert from corners.
Courtney Baker-Richardson put the result beyond doubt in the 77th minute, with Town unable to find a way back into the tie.
Simon Weaver named the same starting line-up that defeated Swindon on the opening weekend, though Tyler Frost was left out the squad due to injury.
Alex were also unchanged from their opening day win over Rochdale, with former Burnley youngster Agyei leading the line for Alex Morris' side.
Joe Mattock had Town's first attempt on goal inside the opening ten minutes, though Arthur Okonkwo saved comfortably what would be Harrogate's most threatening moment of the half.
Callum Ainley had been the hosts' biggest threat in the opening half hour, twice firing powerful efforts narrowly off target.
A good piece of goalkeeping from Pete Jameson kept the score level in the 31st minute, Town's stopper preventing Lachlan Brook's effort from nestling in the bottom corner with a one handed save.
Crewe took the lead in the 45th minute when Zac Williams headed a deep corner back towards the six-yard box, where Agyei was waiting to bundle over the line.
A corner again proved Town's undoing minutes later as Mellor headed another deep delivery home at the far post to hoist his side into a two goal lead.
Town made a bright start for the second half with both Luke Armstrong and Jaheim Headley going close, but failing to test Okonkwo.
With 13 minutes to go, Crewe substitute Baker-Richardson ended all hopes of a Harrogate comeback, following up his initial effort saved by Jameson to claim a third for Crewe.
Alex Pattison had a couple of efforts that forced Okonkwo into action in the closing stages as Town were forced to accept a first defeat of the season.
Town: Jameson, Welch-Hayes, Burrell, Mattock, Thomson, Headley, Dooley, Austerfield (Muldoon 69), Pattison, Daly, Armstrong
Subs not used: Oxley, Richards, Ferguson, Horbury, Ilesanmi
Crewe Alexandra: Okonkwo, Mellor, Williams (Colkett 61), McDonald, Offord, Thomas, Ainley (Griffiths 83), Agyei (Leshabela 83), Brook (Baker-Richardson 69), Sambou (Finney 83), Uwakwe
Subs not used: Booth, Sass-Davies
Goals: Agyei 45, Mellor 45+2, Baker-Richardson 77
Booked: Colkett, Uwakwe
Referee: D Bourne