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To be honest there are many mistakes so it's hard to say which is the biggest. One that comes to mind is data analytics. I think we implemented data analytics a lot later than we should have. Ideally you should have it as early as possible so you can make decisions based on data rather than making guesses.

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Too much refinement before commercialization. No product or solution is perfect at the time that you're doing. As long as it's fitting several important pain-point or necessary, you're good to launch.

Do not afraid of failure, especially criticism from your (potential) customer; Start-up that received the most feedback is the most potential to improve to be the domain expect.