Biofilter, an essential water treatment process, is reported to be the harbor of bacterial antibiotic resistance genes (ARGs). Due to the oligotrophic characteristic of source water, filter biofilm is largely influenced by the concentration of organic carbon. The objective of this study was to investigate the effect of organic carbon concentration on shaping bacterial antibiotic resistome in filter biofilm. Our study was based on pilot-scale sand filters, and we investigated the antibiotic resistome using high-throughput qPCR. A total of 180 resistance genes from eight categories of antibiotics were detected in 15 biofilm samples of three sand filters. The results indicated that higher concentration of influent organic carbon led to lower diversity of bacterial community and richness of antibiotic resistance genes (ARGs) in biofilm. We discovered a negative correlation (p ≤ 0.01) between the richness of ARGs and the corresponding TOC level. Moreover, the absolute abundance of ARGs was positively correlated (p ≤ 0.05) with the abundance of 16S rRNA gene and was determined by the organic carbon concentration. Sand filters with gradient influent organic carbon concentration led to the formation of different antibiotic resistomes and canonical correspondence analysis (CCA) indicated that difference in bacterial community composition was likely the main reason behind this difference. We also observed a similar trend in the relative abundance of ARGs, which increased with the depth of sand filters. However, this trend was more pronounced in filters with low organic carbon concentrations. Overall, this study revealed that the organic carbon concentration determined the absolute abundance of ARGs and also shaped the diversity and relative abundance of ARGs in drinking water sand filters. These results may provide new insights into the mechanism of persistent bacterial antibiotic resistance in drinking water treatment.