"Laundry Day" is, quite possibly, the most adorable song ever. Dancing in the laundromat = extreme squeeing on the part of this troper at the sheer cuteness of it all. Damn you, Joss, for ensuring that it will never be a reality!
Plus Billy's adorable little head-dance.
For me, it's when Billy brings Penny a frozen yogurt and pretends it was accidentally given to him. "What a crazy, random happenstance!" Awwwwww.
Even though it's mixed with a little sadness, in the middle of So They Say we see that even though Billy isn't at the laundromat because he's busy plotting evilly, Penny has brought a frozen yogurt for him.
It's also sweet in So They Say that Moist worries about Dr. Horrible when he throws himself into his work and Moist can't get into contact with him.
Penny's part of My Eyes / On the Rise. While Billy's part is dark ("I cannot believe my eyes, how the world is filled with filth and lies."), hers is uplifting and hopeful ("And you believe there's good in everybody's heart, keep it safe and sound. With hope you can do you part, to turn a life around.")
Subtle and downplayed, and possibly just my imagination, but when Dr Horrible switches the tone of "Slipping" he sends the audience scattering even though during the first half of the song he had them captivated/paralysed with fear. Yet he chooses to make them scatter by firing his Death Ray into the ceiling and be incredibly threatening, telling them to go do things they could quite easily do after watching him kill Captain Hammer. The only reasons I can think of for why he does this is just to protect them in case something goes wrong or to prevent them from watching their hero die. It seems he always chooses to do things where as few people will get hurt as possible. This is most likely why he made the Freeze Ray, why he remote controlled the van along a path where he could keep it in view at all times (considering how he acts during the whole scene), and why he refused to fight Johnny Snow in the park.