The effects of streptomycin (STM) on the development of antibiotic resistance in an aerobic-biofilm reactor was explored by stepwise increases in STM doses (0-50 mg L-1), over a period of 618 days. Totally 191 bacterial isolates affiliated with 90 different species were harvested from the reactor exposed to six STM exposures. Gammaproteobacteria (20-31.8%), Bacilli (20-35.7%), Betaproteobacteria (4.5-21%) and Actinobacteria (0-18.2%) were dominant, and their diversity was not affected over the whole period. Thirteen dominant isolates from each STM exposures (78 isolates) were applied to determine their resistance prevalence against eight classes of antibiotics. Increased STM resistance (53.8-69.2%) and multi-drug resistance (MDR) (46.2-61.5%) were observed in the STM exposures (0.1-50 mg L-1), compared to exposure without STM (15.3 and 0%, respectively). Based on their variable minimum inhibitory concentration results, 40 differentiated isolates from various STM exposures were selected to check the prevalence of nine aminoglycoside resistance genes (aac(3)-II, aacA4, aadA, aadB, aadE, aphA1, aphA2, strA and strB) and two class I integron genes (3'-CS and IntI). STM resistance genes (aadA, strA and strB), a non-STM resistance gene (aacA4) and integron genes (3'-CS and Int1) were distributed widely in all STM exposures, compared to the exposure without STM. This new culture-based stepwise increasing antibiotic approach reveals that biological systems treating wastewater with lower STM dose (0.1 mg L-1) could lead to notably increased levels of STM resistance, MDR, and resistant gene determinants, which were sustainable even under higher STM doses (> 25 mg L-1).