A 60‐day indoor trial was conducted to study the effect of periphyton supplementation on metabolic and immune responses in tiger shrimp, Penaeus monodon. Periphyton developed over bamboo substrate in outdoor tanks (15 m2) was used as dietary supplement for P. monodon (2.02 ± 0.04 g) reared in 1000 L FRP tanks. Graded levels of periphyton were included in shrimp basal diets: 0% (P0), 3% (P3), 6% (P6), 9% (P9) and P0 diet with natural periphyton (NP) over bamboo substrate. At the end of the trial, P6 and NP showed significantly higher (P < 0.01) growth rate, 23.9% and 20%, respectively, compared with control, P0. Comparatively, lower level of metabolic enzymes, such as lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase and alanine aminotransferase, was recorded in treatments P3, P6 and NP compared with control, P0. The periphyton‐supplemented group, P3 had significantly higher (P < 0.05) superoxide dismutase (15.83 ± 0.96) and catalase activity (15.73 ± 0.69) compared to 6.88 ± 2.84 and 9.15 ± 0.67 unit mg−1 protein min−1, respectively, in P0. Similarly, higher total haemocyte counts, 32.58 ± 1.30, 28.51 ± 3.12 and 27.26 ± 4.43 × 106 cells mL−1, were recorded in P6, NP and P3, respectively, compared to P0, 23.57 ± 1.80 × 106 cells mL−1. After challenge with Vibrio harveyi, P3 recorded the highest relative percentage survival 67% followed by NP (58%) and P6 (42%) compared with control. However, treatment with highest periphyton inclusion (P9) did not differ significantly with P0 on growth and immunological parameters. This study indicates that periphyton supplementation at 3–6% level improves growth, immune response and metabolic activities in P. monodon.