At the heigh of the Holocaust in September 1943, Niccacci was the Father Guardian of the Franciscan Monastery of San Damiano in Assisi, Italy. Realizing his ability to help save lives of the persecuted, Padre Rufino helped to provide Jews with false identity papers and gave them protection and sanctuary in the monasteries and convents under his direction.
Together with Don Aldo Brunacci, Bishop Giuseppe Placido Nicolini, and the printers Luigi and Trento Brizi (who falsified the ID and ration cards of the hidden Jewish refugees), Padre Rufino was able to save approximately 300 Jews from the slaughter of the Nazi's.
After the war, Niccacci established a small settlement for destitute Christian and Jewish families in Montenero, outside of Assisi, and served as a parish priest in his home town of Deruta, Italy.
In April 1974, the Yad Vashem Holocaust Memorial Museum in Israel named Niccacci as one of the Righteous among Nations - those Gentiles, like the famous Oskar Schindler, who worked to save Jews during the Holocaust.
On April 11, 1983, President Ronald Reagan, in remarks to the American Gathering of Jewish Holocaust Survivors, said:
"The picturesque town of Assisi, Italy, sheltered and protected 300 Jews. Father Rufino Niccacci organized the effort, hiding people in his monastery and in the homes of parishioners. A slip of the tongue by a single informant could have condemned the entire village to the camps, yet they did not yield."
Niccacci was a subject and the narrator of The Assisi Underground, a book written in 1978 by Alexander Ramati about Assisi's efforts to save Jewish refugees. In 1985, the book was made into a movie of the same title.