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Layer-by-Layer (LbL) technology recently turned out to be a versatile tool for the encapsulation of bioactive entities. In this study, the factual potential of this technology to encapsulate synthetically valuable biocatalysts, that is enzymes and whole cells expressing a specific catalytic activity, was investigated. The biocatalysts were embedded into a polyelectrolyte multilayer system involving poly(allylamine) hydrochloride (PAH) and poly(styrene sulfonate) sodium salt (PSS). The enzymes were adsorbed to CaCO3 or DEAE-cellulose previous to encapsulation. A slight increase (32%) of the catalytic performance was observed for lipase B from Candida antarctica when four layers of polyelectrolytes were applied. On the whole, however, the residual activity of the investigated enzymes after encapsulation was rather low. Similar results were obtained with whole-cell biocatalysts. It was found that the activity decrease can be attributed to mass transfer restrictions as well as direct interactions between polyelectrolytes and catalytically active molecules. Both effects need to be understood in more detail before LbL technology can be advanced to technically efficient biocatalysis.