The one-pot synthesis of 1,4-naphthoquinones by the Diels–Alder reaction of dienes with para-quinones generated in situ with laccase (EC 1.10.3.2, p-diphenol:dioxygen oxidoreductase) in an aqueous medium was developed in this study. The para-quinones were generated in situ by the laccase oxidation of the corresponding 1,4-hydroquinones and subsequently underwent the Diels–Alder reaction with dienes, and further oxidation to finally generate 1,4-naphthoquinones, in good yields. This reaction methodology provides unique green chemistry synthesis for isolation of the naphthoquinones, and without relying on organic solvents or hazardous heavy metal reagents. In this paper, the effects of laccase dose, temperature, and substrate sensitivity on the overall reaction were investigated.