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Fernando de Noronha is an oceanic archipelago in Brazil that has been subjected to major alterations in its natural habitat, as it is exposed to increasing rates of tourism. This research aimed at performing the first survey of spider species on the main island, focusing on the environmental occupation and conservation status of local species. Spiders were sampled through pitfall traps, beating sheets, and active collection in the dry (October 2005) and rainy (April 2006) seasons in several parts of the island, such as urban and protected areas. A total of 1,532 adult spiders from 44 species distributed in 20 families were collected. Forty-two species are newly recorded on the archipelago, of which 10 appear to be native. Theridiidae and Salticidae were the richest families, with seven and five species respectively. Hogna sp., probably an endemic Lycosidae species, had the highest abundance throughout the study (17%). Several non-native species were found, especially in the surroundings of human habitations. Areas exposed to human settlements had higher diversity indices and evenness values when compared to preserved areas. Most species were classified as being diurnal space web-weavers. The results suggest that non-native species seemed to be established on the island, due mainly to the traffic of people and goods from the continent.