Generally, the macrolide antibiotic Tylosin has been considered inhibiting the chemical oxygen demand (COD) removal in anaerobic digestion. In addition, some studies also demonstrated that the Tylosin affected the microbial populations involved during the degradation of organic compounds. Accordingly, the present study investigates the impact of Tylosin to an up-flow anaerobic stage reactor (UASR) performance and archaea populations at reduced hydraulic retention time (HRT), (4–1 day). Results showed only a minor reduction in COD removal efficiency (15%) was observed when the reactor was operated at high feed flow rate (1 day HRT). The minimal effect of the antibiotic on overall reactor performance confirms that the bacteria were adapted to Tylosin at low HRT (1 day). The fluorescent in situ hybridisation analysis revealed that the decrease in HRT from 4 to 1 day did not appear to cause any change in the archaeal community structure, in any of the UASR reactor stages. The predominance of archaeal cells (68–90%) was obvious in all stages of UASR at each investigated HRT with Methanosaeta (65–99%) dominated Stages 1–4 of UASR for all the HRT investigated.