Abstract: The study investigated the effects of pH on a biological desulfurization reactor's performance, biomass, and microbial structure. Using a bio-trickling filter (BTF), it operated under neutral-alkaline (6.0 < pH < 9.5), mildly acidic (2.6 < pH < 5.5), and extremely acidic (pH < 1.0) conditions, respectively. Maximum H₂S elimination capacities achieved were 246.5, 142.8 and 164.6 g/(m³·h), respectively. The BTF performed best under neutral-alkaline condition due to reduced H₂S gas-liquid mass transfer resistance and increased activity of sulfur-oxidizing bacteria, however, this required significant alkaline reagents, raising costs and complicating the process. In extremely acidic condition, acidophilic sulfur-oxidizing bacteria, Mycobacterium, became dominant (79.2%), and the biofilm possessed high sulfur-oxidizing activity (with specific oxygen uptake rate of 1.35 mg/(g·min)). Non-acid-tolerant microbes were eliminated, preventing reactor clogging and reducing biomass control and pH adjustment costs. Thus, by enriching acidophilic Mycobacterium, BTF can maintain high desulfurization performance under highly acidic conditions, broadening the operational pH range of biological reactors.