Marine snow aggregates represent hotspots of carbon remineralization in the ocean. Various aspects of bacterial dynamics have been investigated on marine snow. To date, extracellular enzymatic activities in aggregates have been measured using small substrate proxies that do not adequately reflect the complexity of biomacromolecules such as polysaccharides, proteins and lipids. To address this issue, we used six structurally distinct, fluorescently labelled polysaccharides to measure enzymatic hydrolysis on aggregates formed with a roller table and in aggregate-free (ambient) seawater from two near-coast sites, north-eastern Gulf of Mexico. A single polysaccharide was incubated in aggregates and ambient seawater. Changes in polysaccharide molecular weight were monitored over time to measure the course of enzymatic hydrolysis. All six polysaccharides were hydrolysed in aggregates, indicating a broad range of enzyme activities in aggregate-associated bacteria. Four substrates were also hydrolysed in ambient waters. Epifluorescence microscopy revealed that nearly all of the bacteria present in original waters were incorporated into aggregates. Therefore hydrolytic activities in ambient waters were presumably due to enzymes spatially disconnected from cells and aggregates. Our results show substantial enzymatic activity in cell/aggregate-free seawater, suggesting a significant role of free enzymes in hydrolytic activity in waters from the north-eastern Gulf of Mexico.