Biofilms are complex communities of multiple microbial species which are attached to surfaces or with interfaces. Such beneficial biofilms can be developed in vitro and be used as Biofilm biofertilizer (BFBF). Agro-ecosystems consisting of soil, plant and microbial communities are affected by farmers’ practices (eg excessive use of chemical fertilizers, CF), disrupting its natural cycles through depleting beneficial microorganisms. BFBF application to a crop soil increases N2 fixing bacteria, P-solubilizing fungi and many other useful microorganisms which help reinstate the degraded networks in the agro-ecosystem by replacing lost organisms and also producing various biochemicals such as phytohormones, signaling molecules etc. Thus, the present study was designed for analyzing soil, plant and microbial parameters with the application of farmers’ CF practice and BFBF practice in 25 different locations using rice (Oryza sativa L.) as the test crop, and to reveal the effect of BFBF in re-establishing networks in the agro-ecosystem. Results indicated that parameters were variable among different locations; however there was an increasing trend of rice grain yield in the BFBF practice. BFBF application has improved rhizosphere soil organic matter through microbial action, which has in turn increased rice grain yield over CF alone application in the farmers’ practice. It helped in cutting down chemical fertilizers use up to 50% of the farmers’ practice without hampering yields. This has been attributed to the positive effects of the biofilms in the BFBF practice towards soil and microbial properties. Therefore, it is concluded from this study that the BFBF practice can be considered as an eco-friendly and economically viable method to replace current farmers’ practice of CF alone application.