Wastewater discharged from cheese industries is often characterized by high values of organic pollutants, solids, and nutrients. An aerated trickling biofilter using peat and perlite as filter media was employed in a pilot-scale level in order to evaluate the performance of biofilter for removal of pollutants from cheese industry wastewater. The biofilter was operated for a period of 33 days under laboratory conditions, and several parameters were monitored. The results showed a significant improvement in the quality of treated effluent. The maximum removal efficiencies of chemical oxygen demand and biological oxygen demand were 99.2 and 99.9%, respectively. Significant reduction in total suspended solids (>96%) was also achieved. A stable ammoniacal-nitrogen (NH4-N) removal was accompanied by biofilter. On an average, NH4-N and total nitrogen decreased by 98.7 and 72%, respectively, with a significant portion of NH4-N being converted to nitrate-nitrogen (NO3-N). Also, a molecular approach based on 16S rDNA was employed to analyze the bacterial community composition present in the biofilter. A comparative sequence analysis of excised denaturing gradient gel electrophoresis bands revealed the presence of diverse groups of bacteria belonging to alpha- and beta-Proteobacteria and Bacteroidetes phylum. We conclude from the results that the use of trickling biofilter is highly effective and a potential treatment method for polishing cheese industry wastewater before being discharged into the local environment.