Four Bacillus spp. were isolated from rhizosphere soil of cotton seedlings and identified as Bacillus subtilis, Bacillus sp., Bacillus sp. and Bacillus amyloliquefaciens. Bacillus isolates exhibited antagonistic activities against Fusarium oxysporum and Rhizoctonia solani which were isolated from roots of cotton seedlings showing damping-off symptoms. Greenhouse and field experiments with artificially and naturally infected soils with R. solani and F. oxysporum were carried out. Seeds of cotton (Giza 89) treated with the antagonistic bacteria (four Bacillus spp.) just before sowing were cultivated. Untreated seeds were involved as a control. In greenhouse experiment, the antagonistic B. subtilis and B. amyloliquefaciens significantly repressed the disease incidence to the lowest values in both seasons (2022 and 2023) as compared to the untreated control. Also, under field conditions the same Bacillus isolates significantly decreased the disease incidence. Finally, the application of B. subtilis and B. amyloliquefaciens as bio-controllar significantly increased seed cotton yield. Thus, these bacterial bio-controlling agents could be recommended for association in integrated control system of damping-off fungi in cotton and improving quality of cotton yield.