It disappeared from view for a moment, but then could be seen crawling up the right side of the 82-year-old pontiff's face.
When it reached his ear, Benedict gave it a swat. But it didn't go away - it reappeared on the pope's left shoulder and scampered down his robe.
As the pope left the medieval Prague Castle's ornate Spanish Hall, the spider could be seen hanging from a piece of web.
In June, Obama now famously swatted and killed a fly that intruded on an interview for CNBC at the White House.