Phenol is widely used in industrial processes and has microbial toxicity. However, the effects of a phenol spill on the microbial community are not clear. The present study explored the changes of bacterial communities in river and fish pond sediments after a phenol spill. The bacterial richness and diversity in river sediments were lower on day 30 (36 days after the spill) than on day 0, while they increased in fish pond sediments. The structures and functions of bacterial communities in both river and fish pond sediments were changed, and a more dramatical variation was detected in fish pond sediments. In river sediments, Proteobacteria, Chloroflexi, Acidobacteria, Bacteroidetes, and Nitrospirae were the major bacterial phyla, and Chloroflexi was enriched. In fish pond sediments, genera Brevibacillus dominated bacterial communities initially, and bacterial composition showed a dramatic change on day 30. Most predicted metabolism functions, as well as genetic information processing functions of translation, replication, and repair, were enhanced in both river and fish pond sediments, while they showed an opposite change trend for xenobiotic degradation function. This work could strengthen our understanding of the effects of phenol spills on sediment bacterial communities in both lotic and lentic ecosystems.