Anaerobic digestion (AD) has been widely employed for converting various biowastes into renewable energy. However, AD of gentamicin mycelial residues (GMRs, a byproduct of gentamicin production) is limited by ammonia inhibition and antimicrobial resistance risk. Compared to mesophilic AD (MMAD) of GMRs, this study looked into three semicontinuous AD processes, i.e., codigestion with wheat straw, thermophilic digestion (TAcoD), and AD at shortened retention time (RT). Results showed that a stable and safe AD could be achieved under suitable operating conditions. Co-digestion could effectively mitigate the adverse effect of ammonia inhibition. The methane production increased by 35.86% in TAcoD compared to that in MMAD and 43.99% of hazardous waste was reduced in TAcoD. Concerning the antimicrobial resistance of AD system, gentamicin was degraded efficiently and the degradation process was not involved in the expression of antibiotic resistance genes (ARGs) related to modifying enzyme. Effective removal of ARGs under three operating strategies was associated with a higher reduction in bacterial abundance of potential hosts. In addition, the changes in the relevant proteins for transformation and conjugation as predicted by PICRUSt suggested that thermophilic condition and shorter RT were conducive to the reduction of the dissemination risks of ARGs.