The physics collection consists of 1,550 collection objects that come from the 18th to 20th centuries. The first physics instruments appeared in the museum's collection in 1911. During the 1950s the collection expanded significantly.

The collection mainly contains demonstration and teaching aids from the following fields: mechanics, acoustics, optics, thermal, electricity and magnetism. These aids were used in physics study rooms at schools and monasteries.

Other parts of the collection consist of practical-use instruments: observing (microscopes), measuring (meteorological instruments, weights and measurements (polarographs, spectrometers) and detection (dosimeters). A smaller part of the collection includes experimental instruments (laser, crystalliser, participles detector) coming from research institutes of the Czech Academy of Sciences and from CERN.

Among most significant instruments are static electric machines, the collection of microscopes, barometers, sets of weights (Augsburg, ca. 1725) and meteorological instruments from the observatory at the Prague Klementinum (from the mid 18th to early 20th century). The collection also has several Czech inventions, including the first polarograph created by Nobel-Prize winner Professor Jaroslav Heyrovský in 1924 and prototypes of equipment for manufacturing contact lenses by Otto Wichterle from the 1960s. The first prototype of a Czechoslovak laser also comes from the first period. The collection also features the module for an elementary particles detector from CERN (European Laboratory for Particle Physics) in Geneva from the 1990s.