In this study, an attempt has been made to assess whether a chronic exposure to metals in habitats under a strong industrial pressure might have equipped spiders with biochemical defensive mechanisms enabling them to survive an additional chemical stress. To check this, non-web-building wolf spiders Pardosa lugubris (Lycosidae) and funnel web Agelena labyrinthica (Agelenidae) were collected at five variously polluted meadows and, under laboratory conditions, intoxicated with either single or multiple dose of dimethoate (OP pesticide). Then the activities of detoxifying (carboxylesterase: CarE, glutathione S-transferase: GST), antioxidative (selene-dependent and selene-independent glutathione peroxidases: GPOX and GSTPx) enzymes as well as acetylcholinesterase as a biomarker of exposure to OP pesticides were measured. In web-building A. labyrinthica, even a single application of the pesticide caused the inhibition of CarE, GSTPx and GPOX in individuals from less polluted sites and AChE and GST in specimens pre-exposed to high metal concentrations. Multiple intoxication, irrespectively of the site, caused significant, in comparison to controls, decrease in CarE, AChE and GSTPx activities. Actively hunting P. lugubris seem more resistant to acute pesticide intoxication, since the spiders from each site had a constant level of GST and AChE. In individuals of this species from heavily polluted sites, the inhibition caused by multiple intoxication with dimethoate was stated only for glutathione peroxidases.