Biotrickling filters (BTFs) for hydrophobic chlorobenzene (CB) purification are limited by mass transfer and biodegradation. The CB mass transfer rate could be improved by 150 mg/L rhamnolipids. This study evaluated the combined use of Fe3+ and Zn2+ to enhance biodegradation in a BTF over 35 day. The effects of these trace elements were analysed under different inlet concentrations (250, 600, 900, and 1200 mg/L) and empty bed residence times (EBRTs; 60, 45, and 32 sec). Batch experiments showed that the promoting effects of Fe3+/Zn2+ on microbial growth and metabolism were highest for 3 mg/L Fe3+ and 2 mg/L Zn2+, followed by 2 mg/L Zn2+, and lowest at 3 mg/L Fe3+. Compared to BTF in the absence of Fe3+ and Zn2+, the average CB elimination capacity and removal efficiency in the presence of Fe3+ and Zn2+ increased from 61.54 to 65.79 g/(m3⋅hr) and from 80.93% to 89.37%, respectively, at an EBRT of 60 sec. The average removal efficiency at EBRTs of 60, 45, and 32 sec increased by 2.89%, 5.63%, and 11.61%, respectively. The chemical composition (proteins (PN), polysaccharides (PS)) and functional groups of the biofilm were analysed at 60, 81, and 95 day. Fe3+ and Zn2+ significantly enhanced PN and PS secretion, which may have promoted CB adsorption and biodegradation. High-throughput sequencing revealed the promoting effect of Fe3+ and Zn2+ on bacterial populations. The combination of Fe3+ and Zn2+ with rhamnolipids was an efficient method for improving CB biodegradation in BTFs.