The variability in zooplankton density and species composition in response to tidal oscillations were investigated in the lesser saline upper reaches (E1) and higher saline lower reaches (E2) of the Mahanadi Estuary, located at the mouth of the third largest river in Indian Peninsula. This is the first of its kind high frequency observation over the tidal cycle ranging from highest high tide to lowest low tide in this estuary revealing dynamic variability of zooplankton assemblages. Zooplankton abundance was higher during high tide in comparison to low tide, irrespective of salinity regimes. On the diurnal scale, it was higher at night in comparison to the day at both E1 and E2. The higher abundance of zooplankton groups such as Copepoda, Cladocera, and planktonic larvae during the night at E1 as well as E2 indicating an upward migration. Many of the zooplankton taxa (e.g., Pseudodiaptomus serricaudatus, Pseudodiaptomus sp., Acartia danae, Acrocalanus longicornis, Oithona sp., Corycaeus andrewsi) migrated towards E1 due to tidal effect during high tide and maintained their position even during low tide. In contrast, the prevalence of limnetic taxa (e.g., Brachionus rubens, Polyarthra vulgaris, Bosminopsis deitersi, Moina micrura, Heliodiaptomus sp.) at E2 during low tide indicated a predominant riverine source. The tidal variability of Brachyura (zoea and megalopa) revealed different emergence times that indicated dispersal of zoeas to the adjacent Bay of Bengal and the return of magalopa to the Mahanadi Estuary. Species diversity index was higher during high tide, and prominent at E2. At E1, marine, fresh, marine-brackish, and marine-brackish-fresh zooplankton species dominated during high tide, while brackish-fresh taxa dominated during low tide. Differently, E2 was enriched with marine, marine-brackish, and marine-brackish-fresh taxa during high tide, whereas fresh, brackish-fresh, and marine-fresh dominated during low tide. Salinity and suspended matter influenced the dominant zooplankton taxa at E2 and E1, respectively. Zooplankton assemblages exhibited a pattern of prominent diurnal-spatial variation in comparison to the tidal scale in the Mahanadi Estuary. Overall, this study documented a very high zooplankton diversity (92 taxa belonging to 13 groups) and significant variations in species abundance which highlighted the importance of carrying out sampling over the tidal cycle at contrasting salinity regimes.