While phylogenetic studies have shown covariation between the properties of spider major ampullate (MA) silk and web building, both spider webs and silks are highly plastic so we cannot be sure whether these traits functionally co-vary or just vary across environments that the spiders occupy. Since MaSp2-like proteins provide MA silk with greater extensibility, their presence is considered necessary for spider webs to effectively capture prey. Wolf spiders (Lycosidae) are predominantly non-web building, but a select few species build webs. We accordingly collected MA silk from two web building and six non-web building species found in semi-rural ecosystems in Uruguay to test whether the presence of MaSp2-like proteins (indicated by amino acid composition), silk mechanical properties, and silk nanostructures, were associated with web building across the group. The web building and non-web building species were from disparate subfamilies so we estimated a genetic phylogeny to perform appropriate comparisons. For all of the properties measured we found differences between web building and non-web building species. A phylogenetic regression model confirmed that web building and not phylogenetic inertia influences silk properties. Our study definitively showed an ecological influence over spider silk properties. We expect that the presence of the MaSp2-like proteins and the subsequent nanostructures improves the mechanical performance of silks within the webs. Our study furthers our understanding of spider web and silk co-evolution and the ecological implications of spider silk properties.