The invertebrate fonds is divided into two sections, malacological and arachrological, according to the most numerously represented groups of invertebrates. The largest sub-collection, the evertebratological, consists of molluscs (Mollusca), anthozoans (Anthozoa), spiders (Araneae), crustaceans (Crustacea), annelids (Annelida) and echinoderms (Echinodermata). The invertebrate collection also holds preserved specimens of Myriapoda, other arachnids (Arachnida), flatworms (Platyhelminthes), nematodes and nematomorpha (Nematoida), and the representatives of small phylum and protista (protozoans). These extensive collections are studied by domestic and foreign researchers, students and all who are professionally interested in zoological issues. They are a source for the popularization of zoology, ecology and scientific knowledge about natural development.

The marine invertebrates, specifically sponges (Porifera), Cnidaria, decapods (Decapoda, of which the collection of crabs is the largest) and echinoderms (Echinodermata, represented primarily by starfish and sea urchins), make up a special part of the collection. Marine specimens, acquired primarily via donations from travellers, in particular divers, are the most used for exhibitions, due to their attractiveness.

The most important and scientifically valuable part of the invertebrate collection is the type material, meaning specimens on the basis of which new species are identified and described. Most of the type material relate to spiders (Araneae) and harvesters (Opiliones); next are molluscs (Mollusca), earthworms (Opisthopora), mites and ticks (Acari), centipedes (Chilopoda), scorpions and protista. There are several specimens of type material in the collection of crustaceans (Crustacea), nematomorpha, tardigrades (Tardigrada), rotifers (Rotifera) and millipedes (Diplopoda).

During its 200 years of existence, the malacology section of the Zoological Department of the National Museum (curator: Jaroslav Hlaváč) has gathered a large collection of all groups of molluscs, cnidaria and sponges. It contains not only material gathered in the former Czechoslovakia (the collection of J. Brabenec), but also material from various other parts of the world. This material is acquired through donations, purchases and also by the research of the Zoological department. Among the most valuable is the fond of anthozoans, and, with regard to the molluscs, the specimens of the families of Cypraeidae (Cypraea auratinum), Pleurotomariidae (Pleurotomaria beyrichii) and Conidae; and also typological material. The current state of the malacological collection is at least one and half million items, of which only a tiny fraction are part of the permanent invertebrate exhibition.

The arachnological collection (curator: Petr Dolejš), i.e. spiders and related species, makes one of the most significant parts of the invertebrate fonds. Its principal parts are the collection of spiders (Araneae) of Prof. František Miller and Prof. Jan Buchar and the rich documentation material acquired by Dr. Antonín Kůrka between 1971 and 2011 in connection with faunistic research into the endangered species and protected areas of the Czech Republic. A further important part is František Kovařík’s collection of trapdoor spiders, often used for exhibition purposes. Another large collection is that of harvestmen (Opiliones), which is the result of the life-long scientific work of Dr. Vladimír Šilhavý, and there is also the collection of mites and ticks (Acari) of Doc. Miroslav Kunst. In addition to arachnids, the collection of isopods (Isopoda) of Dr. Marie Flasarová is of significance. The arachnological section includes a large collection of annelid worms (Clitellata), with microscopic slides and fluid-preserved species acquired by collecting in-the-field and through the life-long work of Prof. Sergej Hrabě and Dr. Eva Lišková.