[Background] At present, antibiotics are mainly used to control pathogenic bacteria such as Vibrio harveyi in seawater breeding industry. However, the long-term application or abuse of antibiotics may be adverse to the environment protection and human health. Therefore, biological control methods that are both environmentally friendly and effective have broad application prospects. [Objective] Strains with antibacterial activity were screened from the symbiotic microorganisms of seawater products. The active strains were identified and the culture conditions for the synthesis of antibacterial active substances were determined. [Methods] Fungi and bacteria were isolated from mariculture animals by the dilution and spreading plate method using the culture medium of Sabouraud and 2216E medium. The antibacterial activity of microbial fermentation broths against V. harveyi was determined by Oxford cup method. The active strain was identified by the morphological characteristics and internal transcribed spacer (ITS) analysis. Culture conditions were determined by screening the type and salinity of fermentation medium. [Results] A total of 52 microbial strains were isolated from 9 kinds of samples, among them, 30 strains belong to fungi and 22 strains belong to bacteria. Two fungal strains with antibacterial activity against V. harveyi were obtained. One active strain, HLZ-3, was identified as Aspergillus tubingensi. The culture conditions of HLZ-3 for the synthesis of antibacterial active substances was 4% NaCl in rice culture medium, and stationary incubated at 28 °C for 2 weeks. [Conclusion] The results provide a basis for further isolation and purification of antibacterial secondary metabolites produced by the strain HLZ-3.