It isn't your problem and you aren't the asshole regardless of what you choose. At the same time, no one here can tell you what you should do, in terms of your future happiness. Would locking them out of your room be solely a matter or principle and well-deserved revenge or would it be because you're not sure that you can trust them with the space, the belongings, and the possible squatter's rights? In the case that you trust them with the space and would be punishing them (a totally valid choice) rather than protecting yourself from a legit fear of them abusing your kindness, you would be absolutely justified, but also justified should you choose to prize your relationship with your best friend above your very warranted loathing of his parents.

I am not for one moment telling you that you have the obligation to rise above and make peace, not for one second. I'd cheer you on for drawing that boundary, if it was what you truly wanted. But, if for some reason you value your friendship with this person and also trust that his parents, while annoying, are trustworthy in terms of keeping your space and your belongings safe, I would also not chastise you for placing that friendship first. It's the old "would you rather be right or be happy?" quandary. It's entirely possible that these people are toxic and you need to draw the boundary. It's also possible that you know this friend and his family well enough to know that, despite being assholes, they would also be trustworthy, and that it would genuinely be more important to you to protect the friendship rather than draw that boundary right now. If you feel at all unsafe or like it would be a risk to allow them to stay in your room, say no. However, if it's a matter of drawing a boundary in the name of vengeance or pride (both valid causes here, I'm not belittling them), you're entitled to choose for yourself which is more important for your life and your wishes for the future.

Don't let anyone else guilt you or pressure you into a decision that you don't feel is correct. You aren't the asshole, regardless, you've been beyond decent about this. Good luck, don't ever feel guilty for defining a boundary in order to protect yourself, and I hope it all works out for you.