Chemical compounds such as arsenic, mercury and organochlorine pesticides have been extensively used as preventive and curative conservation treatments for cultural and biological collections to protect them from pest and mold infestations. Most of the aforementioned compounds have been classified as carcinogenic, mutagenic and teratogenic and represent a health risk for members of staff exposed to contaminated objects. The present study addresses the internal exposure of 28 museum employees in Museum für Naturkunde Berlin by measuring arsenic species and mercury in urine as well as hexachlorocyclohexane isomers (α-HCH, β-HCH, γ-HCH), hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (4,4′-DDT) and its main metabolite, dichlorodiphenyldichloroethylene (4,4′-DDE), and pentachlorophenol (PCP) in blood serum. This study was carried out in order to assess the internal exposure of Natural History Museum staff members to toxic metals and organochlorine pesticides. During a working week, two blood samples and five urine samples were taken from each participant, involving 8 women and 20 men. Information about work activity and exposure related factors such as dust development through work, use of personal protective equipment, as well as a nutrition diary were obtained through a questionnaire. Information on fish and seafood intakes as well as amalgam fillings was also available. The results of the study showed that the museum staff members had quantified concentrations of arsenic (median of 6.4 μg/l; maximum of 339 μg/l), mercury (median of 0.20 μg/l; max of 2.6 μg/l), β-HCH (median of 0.12 μg/l; max of 0.39 μg/l) and 4,4′-DDT (median of 0.050 μg/l; max of 0.82 μg/l). Despite that all the concentrations were below the established reference values, multivariate regression models were able to show that museum staff members are currently exposed to the aforementioned compounds while handling museum objects. To validate our findings, further studies are required. •Pesticides and toxic metals have been used as conservation treatments in Natural History Museums.•Museum staff is exposed to As, Hg, β-HCH and 4,4′-DDE while handling objects.•Arsenic and mercury concentrations were associated with skin contact to collection objects.•Dust exposure is associated with higher concentrations of β-HCH and mercury.•Use of gloves is protective for 4,4′-DDT exposure.