Already without Mike Wilson and Louis Moss through injury, and with Gus Williams reporting that Danny Taylor has left the club, the Seagulls suffered another massive pre-match blow when Jonathan Royle pulled up with a leg muscle injury in the warm-up.
The Seagulls were forced to play Chris Noone and Mike Lea as an emergency centre back pairing, with Tom Buckley filling in at left back, and were left with four reserve team players on the bench.
Guiseley quickly took advantage of that upheaval with Adam Royles breaking clear to score after five minutes and Oli Johnson headed a second eight minutes later.
The Bay gradually got to grips with things as the half progressed and were close to pulling a goal back before half-time.
Obi Anoruo lifted the ball over advancing Guiseley keeper Steve Drench, but defender Danny Lowe raced back to clear off the line, and Drench then made a good save to tip a James Ellison header from a Marc Williams cross over the bar.
The Bay had no luck in the second half as Anoruo got the ball in the net, after good work by Marc Williams, only for a late flag to rule the goal out for offside, and then Ellison saw his curling free kick rattle the bar and bounce clear with the keeper well beaten.
The Bay also had a strong penalty shout turned down as Williams went down in the box and Hopley appeared to be pulled back by his shirt.
Yet, after all that, Guiseley broke out and went 3-0 up with an excellent 20-yard free kick into the corner of the net by Wayne Brooksby on 73 minutes.
That was a killer blow, and although the Bay immediately pulled a goal back through Anoruo, Guiseley saw out the game to maintain their push for the play-offs.
Gus Williams admitted his side had paid for conceding two soft goals early on as the new back four were still adjusting to the changes, but added: “After that we had the better chances and had more shots at goal than them, but nothing really went for us in their box.”
The Bay, however, stay ahead of Leamington and Brackley, who both lost, and Williams added: “Our injuries have come at the worst possible time for us, but we have to keep battling because the teams below us are going to be feeling the pressure a lot more with games running out.”
Colwyn Bay: Sanna, Baynes, Lea, Noone, Buckley, Holsgrove, Anoruo, Haywood, Hopley, Williams (c), Ellison. Subs not used: Rees, Saiel, Seddon, Hodgson, Bakker.