Biofloc technology aims to maximize fish farming productivity by effectively breaking down ammonia and nitrite, promoting healthy flocculation, and enhancing the growth and immunity of cultured animals. However, a major limitation in this field is the suitable starter microbial culture and narrow number of fish species that have been tested with the biofloc system. Here, we investigated various microbial inoculum containing beneficial microbes with probiotics, immunostimulatory and flocs development and bioremediation properties would lead to the development of ideal biofloc development. Three treatment groups with different microbial combinations, viz., group 1 [Bacillus subtilis(AN1) +Pseudomonas putida(PB3) +Saccharomyces cerevisiae(ATCC-2601)], group 2 [B. subtilis(AN2) +P. fluorescens(PC3) +S. cerevisiae(ATCC-2601)] and group 3 [B. subtilis(AN3) +P. aeruginosa(PA2) +S. cerevisiae(ATCC-2601)] were used and compared with the positive control (pond water without microbial inoculums) and negative control (clear water: without microbial inoculums and carbon sources) on biofloc development and its characteristic features to improve the water quality and growth of fish. We demonstrated that microbial inoculums, especially group 2, significantly improve the water quality and microbiota of flocs and gut of the test animal,Heteropneustes fossilis. The study further demonstrates that biofloc system supplemented with microbial inoculums positively regulates gut histomorphology and growth performance, as evidenced by improved villous morphology, amylase, protease and lipase activity, weight gain, FCR, T3, T4 and IGF1 levels. The inoculums induced an antioxidative response marked by significantly higher values of catalase (CAT) and superoxide dismutase (SOD) activity. Furthermore, the supplementation of microbial inoculums enhances both specific and non-specific immune responses and significantly elevated levels of immune genes (transferrin, interleukin-1β and C3), and IgM was recorded. This study provides a proof-of-concept approach for assessing microbial inoculums on fish species that can be further utilized to develop biofloc technology for use in sustainable aquaculture.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-023-02107-0.