The collection comprises around 3,300 early printed books (from the Latin "incunabula" = diaper, cradle). In addition to the two Gutenberg Bibles, the collection also includes copies of Hartmann Schedel's famous "Weltchronik" (World Chronicle, 1493) and the "Cosmographia" (1482), a printed atlas based on Ptolemy, which is considered a milestone in map printing. One of the most beautiful works of the 15th century is also represented in the collection. Francesco Colonna's "Hypnerotomachia Poliphili" (The Dream of Poliphilus) from 1492, with woodcuts in the style of the Italian Renaissance, is considered a masterpiece of early book printing.

The collections from the 16th to 18th centuries trace the thematic and media-historical diversity and development of the book. Highlights of the collection include Sebastian Brandt's "Narrenschiff" (Ship of Fools, 1506) with woodcuts, some of which were probably created by the young Albrecht Dürer, and the romantic knightly romance "Theuerdank" (1517), with which the young Emperor Maximilian I immortalized himself. Important scientific works are also part of the collection, such as the "Astronomicum Caesareum," an astronomical book for Emperor Charles V, which Peter Apian printed in Ingolstadt in 1540.

The collection on the history of books from the 19th to the 21st century covers the spectrum from industrial mass production to artistically sophisticated book editions, which were often published in small presses and print runs. Significant exhibits include works by outstanding Art Nouveau type designers such as Otto Eckmann and Rudolf Koch, as well as illustrators including Marcus Behmer and Thomas Theodor Hein. Type samples and type sample books from the most important type foundries and the most significant type designers illustrate the developments in typography.

The Gutenberg Museum's collection includes around 2,500 printed works from the late 19th to the 21st century. These books, which were painstakingly produced using artistic and artisanal techniques, deliberately set themselves apart from the mass-produced "books" that emerged at the beginning of the 20th century as a result of advances in printing technology. Press prints are often characterized by their artistic design. Special attention is paid to every step of the book production process—from font selection to typesetting, printing, and illustration—elevating the book to a "total work of art."

The collection also includes children's books from the 18th to the 20th century. Significant works include Friedrich Justin Bertuch's "Bilderbuch für Kinder" (Picture Book for Children), published in 1790, and Heinrich Hoffmann's "Struwwelpeter" (1844). The collection also includes picture books by famous illustrators of the 19th and 20th centuries such as Kate Greenaway, Arthur Rackham, Ernst Kreidolf, Lieselotte Schwarz, and Maurice Sendak.

Miniature books are defined as books whose book block is no larger than 10x10cm. With the acquisition of Heinz Müller's collection in 2012, the Gutenberg Museum now has the largest collection of miniature books in Europe, comprising around 6,900 items. The collection ranges from fine literature to dictionaries and cookbooks to calendars, offering a unique insight into the world of the smallest books.

The examination of the role of books in modern times has inspired numerous artists to conceive of books as objects that often alienate or negate reading. The Gutenberg Museum's collection of artists' books includes works by Karlheinz Zwick, Julia Farrer, Anton Würth, and Barbara Beisinghoff, among others.

The collection of letter-writing literature comprises over 450 letter-writing guides, i.e. guides to writing correspondence, including valuable incunabula from before 1500. This high-quality collection provides a profound insight into the history and development of letter writing over the past six centuries up to the present day.

The extraordinarily extensive book collection, which contains almost all surviving titles on the genre from German-speaking countries, was compiled by Dr. Erwin Kreim (Mainz) and donated to the Gutenberg Museum in 2011. In its complexity, it provides an overview of public and private communication, which was made possible not least by the invention of Johannes Gutenberg.

Today, the term "letter writer" is largely unknown, although printed instructions on letter writing were once very popular. Anatol Paul Broyard (1920-1990), editor of the New York Times, commented: "In an age like ours, which is not inclined to letter writing, we easily forget what role it once played in people's lives."