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The present research is focused on the application of glass beads (GBs) in fixed biofilm reactor (FBR) for the treatment of simulated methylene blue (MB) wastewater for 9 weeks under aerobic conditions. The COD of MB wastewater showed a reduction of 86.48% from 2000 to 270.4 mg/L, and BOD was declined up to 97.7% from 1095.5 to 25.03 mg/L. A drastic increase in the pH was observed until the 3rd week (8.5 to 8.28), and later, marginal changes between 8.30 ± 0.02 were noticed. A dramatic fluctuation was observed in ammonia concentration which increased (74.25 mg/L) up till the 2nd week, and from the 3rd week it started declining. In the 9th week, the ammonia concentration dropped to 16.5 mg/L. The color intensity increased significantly up till the 2nd week (259,237.46 Pt/Co) of the experiment and started decreasing slowly thereafter. The SEM-EDX analysis has shown the maximum quantity of carbon content in the GBs without biofilm, and then in the GB samples of 1st, and 9th-week old aerobic biofilms. Furthermore, Raman spectroscopy results revealed that the 9th-week GBs has a fine and strong MB peak and matched with that of the MB stock solution. Overall, the results have shown that the GBs filter media were suitable for the development of active biofilm communities for the treatment of dye wastewater. Thus, GBs-FBR system can be used for wastewater treatment to solve the current problem of industrial pollution in many countries and to protect the aquatic environment from dye pollution caused by the textile industry.