Wolf spiders (Lycosidae) are the most abundant ground-hunting spiders in the Australian cotton (Gossypium hirsutum L.) agroecosystems. These spiders have potential in controlling pest bollworms, Helicoverpa spp. (Lepidoptera: Noctuidae) in minimum-tilled fields. A study was carried out during a wet growing season (2011–2012) in Narrabri, New South Wales, Australia, to determine how different crop rotations and tillage affect wolf spider assemblages in cotton fields. Spider abundance and species richness did not differ significantly between simple plots (no winter crop) and complex plots (cotton—wheat Triticum aestivum L.—vetch Vicia benghalensis L. rotation). However, the wolf spider biodiversity, as expressed by the Shannon—Weaver and Simpson's indices, was significantly higher in complex plots. Higher biodiversity reflected a more even distribution of the most dominant species (Venatrix konei Berland, Hogna crispipes Koch, and Tasmanicosa leuckartii Thorell) and the presence of more rare species in complex plots. T. leuckartii was more abundant in complex plots and appears to be sensitive to farming disturbances, whereas V. konei and H. crispipes were similarly abundant in the two plot types, suggesting higher resilience or recolonizing abilities. The demographic structure of these three species varied through the season, but not between plot types. Environmental variables had a significant effect on spider assemblage, but effects of environment and plot treatment were overshadowed by the seasonal progression of cotton stages. Maintaining a high density and even distribution of wolf spiders that prey on Helicoverpa spp. should be considered as a conservation biological control element when implementing agronomic and pest management strategies.