Seasonal and spatial variations in the composition and relative contribution of the harpacticoid species in the Mondego estuary (western Portugal) were studied based on a monthly sampling along a salinity gradient. These benthic harpacticoids were collected in the water column by means of a 63-µm plankton net. The influence of hydrological parameters (temperature, salinity, oxygen dissolved concentration, pH, turbidity, chlorophyll-a, total suspended solids and nutrient concentrations) was analysed by means of a redundancy data analysis. In addition, this is the first study to provide a checklist of benthic harpacticoids from this estuary. In total, 13 species plus six species not yet identified but known to belong to the genus Canuella, Microsetella, Ectinosoma, Mesochra, Harpacticus and Parapseudoleptomesochra were identified. Copepodites and adults of Euterpina acutifrons and Paronychocamptus nanus were most abundant in this harpacticoid community. While P. nanus dominated in winter and spring at upstream stations, E. acutifrons dominated more downstream. Although this species occurred along the whole salinity gradient, its highest abundances were found in the north arm of the estuary, except in autumn. This distribution may be due to the adaptability of E. acutifrons to different environments and the intermediate position between marine and estuarine conditions assigned to this species. Paraleptastacus cfr. spinicauda showed a relative occurrence of 5–10% in all stations, except at the mouth of the estuary (M). Tachidius discipes was found in low densities in Mondego estuary in spite of the large numbers of copepodites of this species in the southern arm, characterized by a high level of total suspended solids. The northern arm of the estuary was characterized by a higher numerical occurrence of harpacticoid organisms throughout the study years, with the higher densities towards the freshwater part (upstream areas). Canuella sp., Ectinosoma sp. (copepodite), Ectinosoma melaniceps, Leptocaris brevicornis, Phyllognathopus viguieri, Microsetella norvegica (copepodite) and Macrosetella gracilis (copepodite) were considered rare species. Nonetheless harpacticoid species were represented by a higher number of adults in the northern arm, and juveniles in the southern arm.