The National Library's collection includes over 6,5 million volumes and yearly, it acquires about 80 thousand of new titles. It provides services to more than one million users every year, most of whom are university or college students, teachers, academics, scientist, and scholars. Each year, around one million books are lent.

The National Library is one of the oldest public libraries in the Czech Republic with collections of a size far outranking any other library in the country. The value of its collections makes it one of the most important libraries both in Europe and in the world. It acquires, preserves, continuously updates, and provides access to rich collections of both domestic and foreign documents, especially of Bohemica and documents from the social and natural sciences areas. Historical collections are mostly of Czech and European origin. Main part of them consist of Bohemica, too. The core of the manuscript collection is formed by a set of codices donated by Charles IV to Prague University after the foundation of its first college in 1366. In addition, there are also oriental manuscripts and Greek papyri to be found there. The manuscript collection includes a large number of unique pieces as, for example, the Vysehrad Codex from the year 1085, created for the coronation of the first Czech King Vratislav, the Passional of the Abbess Kunhuta from the year 1312, produced in the scriptorium of the Convent of St. George at Prague Castle, the Velislav Pictorial Bible from the first half of the fourteenth century.