Mistakes are not bad things, without them we would not know which way is the right way. I think the problem comes when we, or a company do the wrong thing and don't learn from it, instead continuing down that path.
Walt's biggest mistake: smoking.
TDC's biggest mistake: very hard to say. They've made quite a few initial decisions that were bad for either them or the guests, but they've recovered from almost every one that I can think of, and done so well.
If I had to choose one item/category that is a true mistake, I'd say it's those items that didn't start off as having problems, but now, with time, are and are allowed to remain as such. The old attractions that desperately need a make-over to bring them up to date (for example, the speedway which could be improved to use better technology and reduce pollution.) Then there's the opposite: the elements of attractions that Disney has updated but the updates have had other negative effects: for example, the lighting in Peter Pan is brighter and more efficient, but is now allowing us to be distracted by the ceiling, taking the 'magical' quality away.
Along these lines, customer service is headed downhill. Disney still leads all industries with their training and people policies, but over the years there has been a definite decline in the caring attitudes the employees used to take toward the guests and their jobs. The reasons behind this are many, but the fact remains - it's not what it used to be and it's allowed to stay this way.