Spiders are successful natural enemies of pests occurring throughout the different strata of an agroecosystem. The study of their functional responses can provide information related to the potential effectiveness of different species and guilds on reducing a pest population. However, multiple prey availability may change the functional response of a predator. In this study, the functional responses of three species of spiders in single-prey and multiple-prey experiments were modelled. The spider species Haplodrassus rufipes, Araniella cucurbitina and Synema globosum were chosen as being representatives of ground runners, orb-weavers and ambushers, respectively. Three prey species were selected: a target prey, the Mediterranean fruit fly Ceratitis capitata and two alternate prey species, the flour moth Ephestia kuehniella and the house cricket Acheta domesticus. When the selected target prey C. capitata was supplied in the single-prey experiments, the most and the least efficient spider species were H. rufipes and S. globosum, respectively. However, opposite results were obtained when alternative preys were supplied. Also, A. cucurbitina significantly changed its functional response with the presence of alternative prey species. Considering the prey preference during the multiple-prey experiments, A. domesticus, used as representative of a heavy, long-sized and highly motile prey, was avoided whereas C. capitata, the target pest used as representative of light, small-sized and moderately motile prey was preferred by the three species of spiders. Ephestia kuehniella, used as representative of light, medium-sized and low motile prey was occasionally consumed. Each guild could include efficient predators against pests according to its hunting strategies and the ecological exploited niches. Orb-weavers could be efficient predators against flying pests; ambushers such as S. globosum could contribute to the reduction of the populations of flower-visiting pests, whereas active ground hunters may also play an important role preying on pests that develop a part or all of its life cycle in the ground. However, further research on feeding behaviour such as prey switching is needed for a better understanding of the effectiveness of spiders as natural enemies.