I currently have guardianship of my niece. She is almost 4, I received guardianship when she was turning 2 but she has always lived with me except for about 4 months of her early life. At the time I received guardianship my sister insisted she had no knowledge of whom the father could be and even signed a legal document stating so. Now that a few yrs have passed she off and on claims she wants custody back and every other boyfriend she claims to be my nieces father. My question is if for some reason one of them really is the father can my guardianship be reversed or will he still have to prove to be in the best interest of my niece?

If you were appointed guardian by the court, the mother would have to prove that termination of the guardian is in your niece's best interest.

If Tom, or Harry can prove that he is the father, he also can ask the court to terminate the guardian and give custody to him. He would have to make the same sort of showing that the mother would have to make.

Guardianship and custody are different legal terms.

To GV70, Could you please explain how the difference?

Best interest standard is applied in child custody cases.
Change of circumstance/s standard is applied in child guardianship cases.

Let me give you an example.
I have guardianship due parents being homeless. They found a home thus there is change of circumstances which leads the guardianship to be reversed..
The same scenario but I have custody. Then the parents must prove that it is in child best interest to remain with them.