A large collection of microorganisms with over 2000 isolates has been established at the Australian Cotton Research Institute for the purpose of identifying potential bio-control agents and developing biological control methods for seedling diseases and Verticillium wilt. These isolates have been collected from within, on and around the roots of cotton plants collected from commercial crops in different cotton growing areas of New South Wales. The culture collection has been screened for in vitro antagonism against the pathogens that cause seedling diseases (Pythium sp., Rhizoctonia sp.), Verticillium wilt (Verticillium dahliae), black root rot (Thielaviopsis basicola), and bacterial stunt (Pseudomonas sp.) of cotton. About 29% of the culture collection inhibited the growth of one or more pathogens. A part of the culture collection was screened in in vivo glasshouse and growth chamber experiments for activity against the seedling disease pathogens and Verticillium dahliae. Several bacterial strains were found to be effective. Cotton seed inoculation with bacterial strains increased seedling survival by up to 80% (compared to the non-treated control). Some bacterial strains significantly reduced the incidence of Verticillium wilt. The proportion of plants with vascular symptoms ranged from 11-30% compared to 64-78% in the non-treated controls. Some bacterial strains which were effective in pot experiments significantly increased seedling survival in field experiments. The addition of nutrients to the inoculum and the use of combinations of bacterial strains as a means of enhancing biocontrol activity need to be investigated