I would say that the mood at the beginning of this scene is pretty comic.
This scene opens with the players (led by Bottom) talking about how they are going to do their play. This gives Bottom a good chance to be a clown. He starts talking about concerns he has about how the play will go. As he does this, he tells a lot of jokes and generally acts silly. For example, he talks about how the ladies will be afraid of the lion. He then says that there is no wildfowl that is more frightening than a lion.
The mood of this scene is comic but also serious, as Shakespeare continues the discussion that threads throughout the play on the relationship between reality and art/imagination. The players, particularly Bottom and Quince, worry about managing the effects of realism, showing an acute, if comic, awareness of audience, and the way audiences invest imaginatively in a drama.
Tellingly, the scene opens with the players arriving in the forest and Quince reacting to it imaginatively:
This green plot shall be our stage, this hawthorn-brake our tiring-house, and we will do it in action as we will do it before the duke.
The makeshift players then worry about the effects the play will have on the audience, especially the effect of Pyramus drawing a sword to kill himself and a lion coming on stage. This is comic, as the actors feel they have to stop the drama repeatedly to keep reassuring people, especially the women, that the stage action is not real. In fact, they are so fumblingly inept that nobody is likely to be fooled for a moment. Further, in their sincere concern, they also show a complete misunderstanding of the relationship between audience and art: audiences want to believe a play is all real; they want to have the bejesus scared out of them by scary scenes.
So we laugh, but we're also invited to think seriously about art and how it functions, foreshadowing Puck's speech at the end, where he invites the audience to make its own decisions as to what extent the play is a "dream." We are drawn as well into the conversation about the relationship between imagination and love, where people fall in love (such as Titania will with Bottom in his ass's head) in ways which seems to bear little relationship to reality and much to the machinations of desire.