The fish assemblage structure in a port area in São Marcos Bay (Amazonic coast) was evaluated based on the spatial and temporal distributions to identify potential changes in response to anthropic pressure increases associated with industrial and port activities in region. The samples were taken between March 2011 and November 2015. The ichthyofauna was represented by a total of 56 species, distributed in 15 orders and 29 families. Captures were dominated by Genyatremus luteus (Bloch, 1790), but Sciades proops (Valenciennes, 1840) was the most representative in terms of biomass. Seasonal distributions of fish assemblage did not reveal significant differences. However, there was a difference between catch sites, abundance, biomass and Shannon diversity index was higher in the Site 1 and evenness in Site 4. The analysis NMDS and the test ANOSIM between months and between sampling sites, based on species composition, revealed a seasonal differentiation associated with the rainy and drought months, as well as spatial differentiation, in function of a depth gradient and hydrodynamics, resulting from greater distance from mangrove areas. The low diversity recorded may be a reflection of port activities that historically occur in the area investigated. However, there was still a maintenance of regional diversity throughout the period under analysis. Thus, temporal and spatial scales become important for the detection and understanding of fish biodiversity in an Amazonian estuary, reflecting, the importance of mangroves for the maintenance of the ichthyofaunistic diversity in the area. In this context, the present study may subsidize possible conservation projects in the area since information of this nature is almost non-existent for estuarine fish from the Maranhão Amazon.