It is a known fact that Mario and Luigi are plumbers.
But when was that defined, and did it ever have any effect on the plot of a game?
They were first shown to be plumbers in the original Mario Bros. game
The original Mario Bros. game, first released in arcades in 1983, was the first game in which Mario and his brother were shown to be plumbers. In fact, the entire premise of the game was about their plumbing, in which they were attempting to eliminate strange creatures in the sewers of New York. During the game, enemies would come out of the pipes at the top of the screen and make their way down to the pipes at the bottom, which they would enter to loop back to the top.
This was a change from Mario’s first appearance in Donkey Kong, where Mario (at the time called Jumpman) was instead a carpenter.
It was a plot point in Mario & Luigi: Superstar Saga
Relatively early on in this RPG for the Game Boy Advance, the Mario Bros. are required to fix the plumbing system in Beanbean Castle, supposedly to prove their identities. Having been explicitly called out in dialogue, this is likely the most well-known instance of the brothers’ profession being relevant to the plot of one of the games.