The emission of hydrophobic Volatile Organic Compounds (VOCs) is a serious environmental issue. Typically, biofilters (BFs) are employed for their treatment, with the potential enhancement of mass transfer through the addition of surfactants. However, disparate results in previous studies have been observed, attributed to uncontrolled conditions during the introduction of surfactants to BFs. Additionally, there has been limited exploration of microbial consortium adaptation to surfactants. To address these gaps, this study followed two approaches. First, the long-term (247 days) removal of cyclohexane was studied in a stirred tank bioreactor (STBR) inoculated with Rhodococcus erythropolys E1 and using Tween 80 at three times the critical micelle concentration (CMC). Second, the short-term (9 days) impact of two (bio)surfactants [Tween 80 (1 × CMC) and Quillaja Saponin (QS, 1 × CMC)] on the removal of cyclohexane, hexane and toluene was investigated in batch tests using three types of inocula: a pure culture of Rhodococcus erythropolys E1 (X0), a microbial consortium adapted to cyclohexane (X1), and a microbial consortium adapted to cyclohexane with Tween 80 (X2). For long-term operation, the addition of Tween 80 at 3 × CMC improved cyclohexane removal efficiency (RE) to 87 ± 1% (elimination capacity, EC = 145 ± 25 mg m−3 h−1, gas residence time, GRT = 20 min, inlet concentration, Cin = 14.9 ± 2.5 ppmv), compared to a RE of 32 ± 9% (EC = 44 ± 8 mg m−3 h−1, GRT = 20 min, Cin = 15.1 ± 0.7 ppmv) under similar conditions without surfactants. For short-term operation, the addition of QS at 1 × CMC significantly increased biomass growth, resulting in lower maximum specific consumption rates for X1 and X2 compared to scenarios without surfactants or 1 × CMC Tween 80. The most abundant genera in X1 and X2 were Paludisphaera (26-23%), 67-14 genus (17–23%), and Rhodococcus (9–18%), respectively.