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Aim To evaluate the interactive effects of oil contamination and chemical dispersant application on bacterial composition and sediment remediation of an estuarine port environment. Methods and Results A multifactorial controlled microcosm experiment was set up using sediment cores retrieved from an estuarine port area located at Ria de Aveiro lagoon (Aveiro, Portugal). An oil spill with and without chemical dispersant addition was simulated. Sediment oil hydrocarbon concentrations and benthic bacterial community structure were evaluated by GC-MS and 16S rRNA high-throughput sequencing respectively. Although initially (first 10 days) chemical dispersion of oil enhanced the concentrations of the heavier polycyclic aromatic hydrocarbons and of the C-22-C-30 alkane group, with time (21 days), no significant differences in hydrocarbon concentrations were detected among treatments. Moreover, no significant changes were detected in the structure of sediment bacterial communities, which mainly consisted of operational taxonomic units related to hydrocarbon-contaminated marine environments. We hypothesize that the environmental background of the sampling site preconditioned the communities' response to additional contamination. Conclusion This experimental microcosm study showed that the chemical dispersion of oil did not influence sediment remediation or bacterial community composition. Significance and Impact of the Study Our study showed that chemical dispersion of oil may not improve the remediation of port sediments. Further studies are needed to investigate the impact of chemical dispersants in combination with bioremediation strategies on the process of sediment remediation in port areas.