Doubt and faith are the underlying theme. Could it be that were also being told to have faith. Faith in ourselves, faith in God, faith and trust in our fellow man? Really, all we have is to trust in God when we have been betrayed. Sister Aloysius doubts that the young teacher is a good teacher and able to control and manage her students. She also feels the young teacher's students are not afraid of her enough.
What is really leaping off the pages here is that there's
doubt all through this play. It is very apropos that this play is called what it is.

 Without any evidence, how can someone accuse a priest of molesting a child when the child doesn't even come forth? Just because they see the priest take the child to the rectory, how does this thought come into her mind? I see a lot of mystery in this play. The Senior Sister even doubts herself that anything will be done if she comes forward with this accusation.
It looks like apart from doubt, the themes in this play are guilt, shame and defeat playing out. Sister Aloysius is a busy-body that's out of tune with reality. It's people like this that falsely accuse someone and have them going to jail for a crime they didn't commit. I like the way Father Flynn says, "Well. If I had judged my conversation with Donald Muller to be of concern to you, Sister, I would have sat you down and talk to you about it. But I did not judge it to be of concern to you." Before it was mentioned, I believed that Sister Aloysius was out for him because for some reason, she did not like him. The truth was really distorted in this play.  The truth was doubted. When Father Flynn tells Sister James, "The most innocent actions can appear sinister to the poisoned mind," I'm reminded of the story of a Pastor that car broke down and he went into a bar because that was the only place that he could make a phone call because there were no cell phones in those days and when he came out of the bar, someone spread rumors in the church and accused him of drinking. Mrs. Mueller goes to tell the Sister that she believes that she has it out for Father Flynn .
What's really the twist was when Mrs. Mueller said let Father Flynn have her son Donald and implied that he may be gay. Mrs. Mueller even go as far to say that the sister does not know for sure that there is a relationship between her son and Father Flynn. Here we see the doubt again. I truly understand the character of the The Antagonist Sister Aloyius. In the end, the Father still gets to work in his profession and he gets a promotion at another church. "What the devil meant for Evil, God turns it around for Good."
Genesis 50:20

The parable seen in this story shows the moral character one should possess, as well as, one of The Commandments that says that, “"We should not bear false witness against thy neighbor." Exodus 20:16
Sister Aloysius Spoke unjustly against Father Flynn to the prejudice of his reputation.