[Background] Cotton plays a key role in Xinjiang’s agriculture, but cotton production and fiber quality are seriously affected by Verticillium dahliae, effective prevention and control approaches are the primary tasks to be solved. [Objective] To study the ability of Bacillus velezensis BHZ-29, Bacillus atrophaeus SHZ-24, Bacillus subtilis SHT-15 and Bacillus vanillea SMT-24 to produce siderophore and siderophore’s effects on antibacterial activity. [Methods] MKB-CAS double-layer plate was used to detect siderophore, Arnow method and iron perchlorate method were used to identify the type of siderophore, CAS dye liquid quantitative siderophore and agar diffusion method was used to determine the effect of siderophore on antibacterial substances under low iron conditions. [Results] An orange-purple color appeared around the bacteria on the MKB-CAS solid plate, Solutions color of the four antagonistic antibiotics in the Arnow experiment was red and perchlorate reaction solutions was yellow. The maximum values of quantitative activity units of siderophore of BHZ-29, SHZ-24, SHT-15 and SMT-24 strains were 8.27%±0.61%, 31.80%±2.06%, 16.06%±3.61%, and 15.53%±0.51%, respectively, the absorbance values of As/Ar the smallest ratios were 0.917±0.092, 0.682±0.021, 0.845±0.005 and 0.846±0.008, respectively. No inhibitory zone appeared in the 0, 10 and 20 μmol/L iron ion treatment groups of BHZ-29, SHZ-24 and SMT-24 strains, in three treatment groups of SHT-15 strains had inhibition zones, but there was no difference in the diameter of the inhibition zone. [Conclusion] The four strains of antagonistic bacteria were all siderophore-producing, the type of siderophore was catechol, and the four antagonistic strains had low siderophore-producing ability, with a maximum of 31.80%±2.06%, when the siderophore-producting capacity of As/Ar ratio was higher than 0.682, BHZ-29, SHZ-24 and SMT-24 fermentation sterile filtrates with 0, 10 and 20 μmol/L iron ions, had no effect on the antibacterial activity, SHT-15 fermentation sterile filtrates with 0, 10 and 20 μmol/L had no significant difference in the effect of antibacterial substances.