I would say that the original special split ("special" in gen I to special attack and special defense in gen II) and the physical/special split in gen IV were probably the most impactful from a competitive perspective. Natures and the revamp of the IV/DV and EV/Stat Exp system from gen II to gen III were pretty significant too. All of these changes had a significant effect on the viability of a lot of Pokemon, and changed the way players approached the game overall. And of course, breeding affected how players could go about getting Pokemon, and what moves they could have, so it ties in with the others to a degree.
Mega evolution influenced the game to a degree, but while it changed how teams were built, it didn't affect the viability of Pokemon overall in the same way that some of the earlier stat changes (special split and physical/special split) did, so I would deem it less significant than those other changes.
In terms of affecting non-competitive players, I would say that global wifi and trading were very important. Suddenly, Pokedex completion didn't depend on knowing friends with the games or buying multiple versions and systems and trading with oneself. For me, the addition of wifi trading really enhanced my involvement in internet forums, and made Pokemon a much more interpersonal experience than it had been for me in gen III (and even in gen I/II, where most of my friends bought the same version of the game and didn't like trading much).