Reading your entire question I see some possible mistakes from you. I infer them from your question so they may not really have happened, but it looks like you did them.
Tell your players about what you want from them.
It can be very straightforward and be a simple discussion with your player about what the campaign should be about, including its tone and what frowned upon (see the Same Page Tool). For example one rule of your campaign can be "you are playing nice guys, if your character turns evil you will have to reroll" or anything you agree with your players.
It can also be done with examples through the game, like a NPC who ask them to kill the bandits but no matter what not kill their leader since he is the only one who know where the McGuffin is, or to spy in the bandit camp but don't confront them directly, they are very powerful and they would kill you. You have plenty of different type of quests and killing someone is rarely the ultimate goal. This is different from building a scenario where at some point the PCs have to spare an opponent to get a precious piece of information since in the examples I provided the PCs are explicitly told about the reason why they should play subtle.
Have consistent and predictable rules
Rules never reflect reality perfectly. They have flaws and everyone around the table should be aware of that. For example in D&D a normal human can carry ten kilos of cotton in his backpack. Does that make sense? No. Is it a problem? Not really, since nobody wants to track the volume of each piece of inventory. "The volume doesn't matter" is a trope in many RPGs and thus PCs won't take volume into consideration when preparing their backpacks, or they will do it but during ellipsis and players will never talk about that. It is completely ok if one of the common tropes of the game you are playing doesn't suit you to rule it out. You can if you want write a houserule to take volume encumbrance into consideration, tell your players about that, and play with it. The problem is that is has to actually be a rule, don't just expect your players to assume the trope is flexible and that when they will have to enter a tight cave they will have to remove their backpack.
I once played a campaign of Teocali (FYK in Teocali killing anyone is always seen as a serious spiritual crime and causes bad stuff, like if in D&D you would take -4 to all your mental rolls until you atone) and in the beginning the GM told me that my bludgeoning mace wouldn't kill anyone (dealing the equivalent of nonlethal damage) unless I specifically ask for it. We played like that for months, and I got used to the habit of taking all the bonus possible when hitting bad guys, until at some point after a particularly powerful blow he told me I just killed my opponent. Of course, it made sense that as my PC was really strong such a heavy blow would kill the NPC, but as until there we always tacitly kept the previous rule of "bludgeoning damage is nonlethal" I was still expecting it to work that way. Hopefully the drama it created was nice to play, but it could also just have rendered my PC unplayable.
For your case you should have told the Paladin before he performed his trample that he couldn't choose to do nonlethal damage, and let him change his action accordingly. If you don't he will probably assume that the rules work the way they worked until now and not necessarily that this attack is different from the previous others.
Don't punish
Punishing is for people who do bad things. Your players are playing a game: this is not a bad thing. You may want to change the way they play it but don't see it as a punishment.