Spiders are common inhabitants of human constructions for various purposes. Premises of museums are poorly studied in terms of synanthropic spiders. In 2018-2022, we examined 12 museum institutions in Odesa, Mykolaiv, and Kherson Regions of Ukraine and revealed spiders belonging to nine genera and eight families. Most of the spiders found are typical indoor dwellers in a temperate climate (Pholcus phalangioides, Scythodes thoracica, Tegenaria domestica, Steatoda triangulosa). Spiders can be an additional source of information about the sanitary and hygienic conditions of the museum premises and possible ways of invertebrate invasions. There is also a risk of developing allergic reactions in museum staff and restorers when working with objects contaminated with the remains of spiders’ vital activity. The harmfulness of spiders to museum pieces is manifested in contaminated surfaces with remains of their vital activity (nets, cocoons, excrements, exoskeleton, dead spiders and their preys), biochemical destruction of its components, and microbial contamination of the surface of artworks. Dead spiders and remains of their vital activity can be used as food, housing and shelter by other museum inhabitants. On the other hand, spider victims are mostly museum pests and permanent residents of museum premises. Occasional species (visitors) made up about a third of their prey. We found that spiders reduced the number of wood pests in the museums: from 20 to 30% of the collected pit-prop beetles (Hexarthrum exiguum) and common furniture beetles (Anobium punctatum) were killed and entwined with cobweb silk. The other museum pests in the spider nests were Anthrenus scrophulariae, Attagenus smirnovi, Stegobium paniceum, and Lasioderma serricorne. In general, the content of spider nets gives additional information about other residents of museum premises.