Enzymatic oligomerization of flavonoids enables the synthesis of biomolecules with different structures and improved physicochemical and biological properties and can therefore broaden their application in industry. In this study, the influence of the key reaction parameters temperature, solvent, substrate, and enzyme concentrations on the synthesis of esculin oligomers was investigated. The reaction was optimized using response surface methodology (RSM) in order to obtain the highest products' concentration and specific products' yield (per mass of enzyme). Mass spectrometry revealed that oligomers with a degree of polymerization of up to 4 were synthesized in which dimers were the most abundant, while the NMR analysis of the esculin dimer product showed that C8-C8 link between two esculin units was formed. Maximum products' concentration was obtained at 60 °C, in 14% (v/v) methanol, 7 mg/mL of esculin, and 54.6 U/L of laccase after 7 h, while the optimal conditions for specific products' yield differed in the aspect of optimal laccase concentration which was 19 U/L for this output. Synthesized esculin oligomers exhibited higher iron chelating and cupric reducing antioxidant capacities and similar or even superior free radical scavenging activity compared to monomeric esculin. Moreover, the mixture of synthesized esculin oligomers has shown a promising potential to be used as a skin prebiotics, suggesting novel applications in skincare industries.