A number of mygalomorph spiders cause bites in Australia, including the funnel-web spiders (Hexathelidae, Atracinae: Hadronyche and Atrax) and mouse spiders (Actinopodidae: Missulena). There is ongoing debate about the significance of bites by mouse spiders and the frequency of severe envenoming by funnel-web spiders. We conducted a prospective cohort study of definite spider bites with expert spider identification and include the analysis of mygalomorph spiders here. Subjects were recruited prospectively from February 1999 to April 2003 from patients presenting to participating hospitals or contacting a state poison information centre. Forty-nine cases of bites by mygalomorph spiders were included: 16 were by funnel-web spiders, 13 by mouse spiders and 20 by other trapdoor spiders (Families Idiopidae and Nemesiidae). Of the 49 bites, 45 (92%) occurred on distal limbs (hands and feet). Local effects included severe pain (53%), puncture marks (61%) and bleeding (27%), local redness (33%). Itchiness did not occur. The following were highly statistically associated with mygalomorph spider bites compared to all other spiders (p<0.0001) circumstances (gardening at the time (likelihood ratio (LR) 7.9) and distal limb bites (LR 2.0)) and early clinical features (presence of puncture marks OR bleeding (2.3), or severe pain (2.0)). Of 16 funnel-web spider bites, there were 10 cases with minor local effects, four with moderate envenoming (non-specific systemic or local neurotoxicity) and two with severe envenoming requiring antivenom. In addition to local effects, mouse spider bites caused local paraesthesia in three cases, local diaphoresis in one case and non-specific systemic effects in five cases, but not severe envenoming. True trapdoor spider bites caused only minor effects. The data from a mixed species sample of funnel-web spiders confirms previous observations suggesting that only a small proportion of funnel-web bites cause severe effects. Mouse spider bites are unlikely to cause major envenoming but the clinical effects are consistent with neurotoxic venom and are more severe than the trapdoor spiders.