As primary drivers of organic matter re-mineralization and trophic carbon transfer, bacteria promote biogeochemical cycling and shape ecosystems globally. We studied bacterial communities spatially and seasonally in an impaired harbour of Lake Ontario by extracting and sequencing community DNA from water samples collected biweekly from different sites. Assembled contigs were annotated at the phylum level, and Cyanobacteria were further characterized at order and species levels. Actinobacteria were most abundant in early summer, while Cyanobacteria were dominant in mid-summer. Microcystis aeruginosa and Limnoraphis robusta were most abundant throughout the sampling period, expanding the documented diversity of Cyanobacteria in Hamilton Harbour. Functional annotations were performed using the MG-RAST pipeline and SEED database, revealing that genes for photosynthesis, nitrogen metabolism and aromatic compound metabolism varied in relative abundances over the season, while phosphorus metabolism remained consistent, suggesting that these genes remain essential despite fluctuating environmental conditions and community succession. We observed seasonal shifts from anoxygenic to oxygenic phototrophy, and from ammonia assimilation to nitrogen fixation, coupled with decreasing heterotrophic bacteria and increasing cyanobacteria relative abundances. Our data contribute important insights into bacterial taxa and functional potentials in Hamilton Harbour, revealing seasonal and spatial dynamics that can be used to inform ongoing remediation efforts.