A synthetic method to obtain α- and β-pyran naphthoquinones 10 and 11 with a hydroxyl substituent on the aromatic ring was developed. Two series of α- and β-pyran naphthoquinones were obtained from the 8-hydroxy-lawsone, and their anticancer properties were evaluated against four tumor cell lines. In general, the new compounds displayed good activity, possibly indicating that these compounds have increased pro-oxidant capacity. The 9-hydroxy-α-lapachone and 7-hydroxy-β-lapachone analogues of the natural products α-lapachone and β-lapachone were successfully produced by this methodology.