Considering the adaptability and responsiveness of microorganisms to environmental changes, their indicator potential is still not acknowledged in European directives. This comprehensive study examined the changes of microbial communities in sediments and a range of geochemical parameters from pristine and anthropogenically impacted coastal areas in the eastern Adriatic Sea. Various analytical methods found evidence of sediment contamination (high toxicity level, enrichments of metals, tributyltin) in certain areas, leading to the categorization of sediments based on the level of anthropogenic disturbance. Prokaryotes were identified as the most promising group of microbes for further research, with specific bacterial families (Rhodobacteraceae, Ectothiorhodospiraceae, Cyclobacteriaceae) and genera (Boseongicola, B2M28, Subgroup 23, Sva0485, Thiogranum) proposed as potential indicators of environmental status. Finally, predictive models were developed to identify key indicator variables for assessing anthropogenic impact in sediments. This research represents an essential step toward incorporating microbial communities into assessments of benthic environmental health.