Mysterium Tremendum was created by having the Mickey Hart Band, with guest percussionists and other musicians, play music along with what Hart has called "cosmic sounds". These sounds were created by taking light, radio waves, and other electromagnetic radiation given off by celestial objects such as the sun, planets, stars, and galaxies, and using computers to transform it into sound waves. Hart stated, "I have combined sonic images of the formation of our universe with sounds drawn from musical instruments. It's all about the vibrations that make up the infinite universe. In this case, they began as light waves and these light waves are still washing over us. Scientists at Penn State, Lawrence Berkeley Labs, and Meyer Sound have transformed these light waves into sound waves." Hart said that he worked with George Smoot, a scientist who, with John C. Mather, was awarded the Nobel Prize in Physics in 2006 for his work in cosmic microwave background radiation.
Seven of the twelve tracks on the album have lyrics written by Robert Hunter, who wrote the words for many Grateful Dead songs.