The darkest hour is a foretold day where the Transformers will face the threat of utter extinction. In that time, it is said that a Prime will rise from the ranks of the Autobots and open the Matrix of Leadership, using its power to "light our darkest hour" and eliminate the threat once and for all.
When Optimus Prime confirmed that destroying the space bridge was necessary, Ratchet affirmed that they would "light [their] darkest hour."
When Megatron and Optimus Prime traveled into Unicron to defeat him, Optimus announced that "the power of the Matrix will light our darkest hour" before opening his chest and unleashing its energies.
When Optimus was restored from his Orion Pax persona and returned to the Autobots, Ratchet said it had been the Autobots' darkest hour without him.
After Optimus acquired the Star Saber, Megatron remarked that the Decepticons faced their darkest hour.
Though no one stated it out loud, it is the episode's name and the Decepticons destroying the Autobot base was probably their darkest hour, anyways.