Michael's biggest regret was most likely killing off Fredo. Had he been kept alive, it can also equally be Michaels' biggest regret.

Fredo is known for being easily used or making mistakes that can cost the Family the empire. Fredo couldn't manage his own life and was a tickiing time bomb waiting to exploed at Michael's face. Fredo became a virus to the family and Michael had to protect it at all costs.