We pre‐grew shrimp under polyculture (shrimp‐tilapia ‘ST’ and shrimp‐mullet ‘SM’) and monoculture (P‐control) conditions until they reached the size of 10–12 g. Then, the shrimps were collected and injected with Vibrio alginolyticus (2.1 × 105 CFU shrimp−1). Some shrimp, originating from the shrimp monoculture, was also injected with sterile saline solution, served as N‐Control. Over 8 days, mortality in N‐control was significantly lower than that in the other treatments (p = 0.001). Total haemocyte counts (THC), phenoloxidase activity (PO) and respiratory burst activity (RB) in haemocytes decreased sharply after injection and were significantly different in P‐control, ST and SM as compared to N‐control (p < 0.05). However, there were no significant differences in shrimp survival and immune parameters among P‐control, ST and SM (p > 0.05). It is concluded that the application of shrimp‐fish polyculture systems does not contribute to the robustness of cultured shrimp as verified by challenge through injection. The second experiment was carried out to verify the effect of shrimp‐mullet polyculture on the prevalence of Vibrio spp. Mullet (3.45 g) were supplemented (either biomass of 500 or 700 g m−3) into six aquaria where shrimp (0.48 g) was stocked at a density of 80 g m−3. The other six aquaria were just stocked with shrimp as P‐control and N‐control. Aquaria were inoculated with V. harveyi at a density of 103 CFU ml−1, except for N‐control. Results showed that the presence of mullet at either biomass of 500 or 700 g m−3 could reduce TCBS counts in water by 20.9% and 24.3%, respectively, as compared to shrimp monoculture.