After starting the season with three successive draws, Darmstadt claimed their first win since returning to the Bundesliga as they continued their unbeaten run with a shock win.
Leverkusen struggled to find their rhythm in possession after the early setback, but Roger Schmidt saw his side improve as the first half progressed.
However, opportunities were difficult to come by for the hosts and they wasted their best chance of the opening 45 minutes when Karim Bellarabi finished a break by firing wide of the post.
The Germany international was a constant threat to the Darmstadt defence and he came close once again early in the second half when Christian Mathenia pushed away a powerful strike.
Despite struggling to make the most of regular counter-attacks, the visitors looked untroubled for long spells as they kept Leverkusen's creative players quiet.
However, a frantic finish followed as Leverkusen came agonisingly close to taking the lead when Kyriakos Papadopoulos somehow managed to send a free header wide.
The introduction of Javier Hernandez added a different threat to the Leverkusen defence and the former Manchester United forward almost handed his new side the lead when his header forced Mathenia into a fine save.
A breakthrough still looked probable heading into the closing stages, but Darmstadt held on for a memorable win after Stefan Kiessling fired a great chance over the crossbar late on.