Soundscape ecology provides a long-term, noninvasive approach to track animal behavior, habitat quality, and community structure over temporal and spatial scales. Using soniferous species as an indicator, biological soundscapes provide information about species and ecosystem health as well as their response and resiliency to potential stressors such as noise pollution. Charleston Harbor, South Carolina, USA provides important estuarine habitat for an abundance of marine life and is one of the busiest and fastest growing container ports in the southeast USA. Six passive acoustic recorders were deployed in the Charleston Harbor from December 2017 to June 2019 to determine biological patterns and human-associated influences on the soundscape. Anthropogenic noise was detected frequently across the estuary, especially along the shipping channel. Despite this anthropogenic noise, biological sound patterns were identified including snapping shrimp snaps (Alpheus spp. and Synalpheus spp.), fish calling and chorusing (Sciaenidae and Batrachoididae families), and bottlenose dolphin vocalizations. Biological response to anthropogenic activity varied among trophic levels, with decreased detection of fish calling when anthropogenic noise occurred and increased dolphin vocalizations in the presence of anthropogenic noise. Statistically, fine-scale, temporal patterns in biological sound were not clearly identified by sound pressure levels (SPLs), until files with anthropogenic noise presence were removed. These findings indicate that SPL patterns may be limited in their interpretation of biological activity for noisy regions and that the overall acoustic signature that we find in more pristine estuaries is lost in Charleston Harbor.