When Dave comes out of the kitchen in the cupcake shop with the minion cupcake, Lucy splits it apart, then pins Dave down and asks Gru if he knows him. But when she captured Gru and took him to the submarine, there were two minions with him and Lucy never asked about them then.
The goof items below may give away important plot points.
When we are first introduced to Eduardo/El Macho, he orders cupcakes with Mexican flags on them. He then opens his shirt to display a tattoo of the Mexican flag. Towards the end of the movie when he his wearing the El Macho villain suit, most of his chest is visible and the tattoo is no longer there.
At the beginning of the film, El Macho's ship first attracts the coins of the two card players. This is impossible as globally, all coins are minted using non-ferrous metals or alloys so as not to be magnetizable.