Orb-weaving spiders use a highly strong, sticky and elastic web to catch their prey. These web properties alone would be enough for the entrapment of prey; however, these spiders may be hiding venomous secrets in the web, which current research is revealing. Here, we provide strong proteotranscriptomic evidence for the presence of toxin/neurotoxin-like proteins, defensins and proteolytic enzymes on the web silk from Nephila clavipes spider. The results from quantitative-based transcriptomics and proteomic approaches showed that silk-producing glands produce an extensive repertoire of toxin/neurotoxin-like proteins, similar to those already reported in spider venoms. Meanwhile, the insect toxicity results demonstrated that these toxic components can be lethal and/or paralytic chemical weapons used for prey capture on the web; and the presence of fatty acids in the web may be responsible mechanism for open the way to the web-toxins for accessing the interior of prey's body, as showed here. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among two spider groups - Araneomorphae and Mygalomorphae; and the findings showed protein sequences similar to toxins found in the taxa Scorpiones and Hymenoptera in addition to Araneae. Overall, these data represent a valuable resource to further investigate other spider web toxin systems; these data also suggest that N. clavipes web is not a passive mechanical trap for prey capture, but it exerts an active role in prey paralysis/killing using a series of neurotoxins.