Aims: The present work aimed to design an optimized medium to yield a higher production of glycosides by Pseudoalteromonas issachenkonii KMM 3549T. Methods and Results: Higher levels of fucoidan hydrolase, alginase, laminaranase and b-N-acetylglucosaminidase production were obtained with peptone concentrations ranging from 2·5 g l-1 to 10 g l-1, while the presence of both yeast extract and glucose did not affect enzyme production. The activity of fucoidan hydrolase and laminaranase increased up to 4·83 µM h-1 mg-1 and 19·23 µM h-1 mg-1 protein, respectively, in growth media containing xylose (1·0 g l-1), laminarin (0·5 g l-1) or alginate (0·5 g l-1), and production of b-N-acetylglucosaminidase substantially increased in the presence of fucoidan (0·5 g l-1) or galactose (1 g l-1). All polysaccharides tested in concentrations of 0·5 g l-1 fucoidan and 0·2 g l-1 fucose induced production of alginase (up to 5·06 µM h-1 mg-1 protein). Conclusions: The production of glycosidases is not only stimulated by the presence of algal polysaccharides, but may also be stimulated by monosaccharides (e.g. xylose). Significance and Impact of the Study: The production of glycosidases by Pseudoalteromonas issachenkonii KMM 3549T was significantly improved by using a simple nutrient medium containing peptone (2·5 g l-1) and xylose (5·0 g l-1) in 100% natural seawater.