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The objective of this research was to evaluate the biodegradation of chloroform by using biotrickling filter (BTF) and determining the dominant bacteria responsible for the degradation. The research was conducted in three phases under anaerobic condition, namely, in the presence of co-metabolite (phase I), in the presence of co-metabolite and surfactant (phase II), and in the presence of surfactant but no co-metabolite (phase III). The results showed that the presence of ethanol as a co-metabolite provided 49% removal efficiency. The equivalent elimination capacity (EC) was 0.13 g/(m(3) h). The addition of Tomadol 25-7 as a surfactant in the nutrient solution increased the removal efficiency of chloroform to 64% with corresponding EC of 0.17 g/(m(3) h). This research also investigated the overall microbial ecology of the BTF utilizing culture-independent gene sequencing alignment of the 16S rRNA allowing identification of isolated species. Taxonomical composition revealed the abundance of betaproteobacteria and deltaproteobacteria with species level of 97%. Azospira oryzae (formally dechlorosoma suillum), Azospira restrica, and Geobacter spp. together with other similar groups were the most valuable bacteria for the degradation of chloroform.