Chalcogen-containing β-lapachone derivatives were synthesized using a straightforward methodology and evaluated against several cancer cell lines (leukaemia, human colon carcinoma, prostate, human metastatic prostate, ovarian, central nervous system and breast), showing, in some cases, IC50 values below 1 μM. The cytotoxic potential of the lapachones evaluated was also assayed using non-tumor cells: human peripheral blood mononuclear cells, two murine fibroblast lines (L929 and V79 cells) and MDCK (canine kidney epithelial cells). These compounds could provide promising new lead derivatives for anticancer drug development. This manuscript reports important findings since few authors have described C-3 substituted β-lapachone with potent antitumor activity. The methodology employed allowed the preparation of the compounds from lapachol within a few minutes in a green approach.