Spider silk is the world's toughest biological material yet very few spider species silks have been characterised. We analysed both the silk gland transcriptome and the silk mechanical properties of the exceptionally large Australian rainforest golden orb weaver Nephila pilipes in comparison to the related garden variety spider Nephila plumipes. Illumina next-generation sequencing of the major ampullate gland coupled with LC-MS/MS of the solid silk fibre identified highly expressed genes within the gland that may be involved in the formation of the silk fibres from the soluble to solid state, as well as proteins that may contribute to stabilization of the fibre. Biomechanical analysis of the two silk threads indicated Nephila pilipes had significantly tougher silk with higher strain capacity than its smaller congener, producing threads with toughness up to 305 MJ/m3. Within N. pilipes, smaller silk fibrils were produced by larger spiders, yielding tougher threads whose fibril diameter was negatively correlated with silk toughness. In contrast, while spider size was correlated with thread diameter in N. plumipes, there were no clear patterns relating to silk toughness, which suggests that the differences in properties between the silk of the two species arise through differing molecular structure.