The first total synthesis of bipenicilisorin (1) isolated from Penicillium chrysogenum SCSIO 41001 via its monomer natural product, penicilisorin (2), was achieved. Penicilisorin was synthesized in four steps from a o-bromobenzaldehyde derivative via the Pd-catalyzed one-pot fluorocarbonylation/lactonization/β-elimination cascade reaction. Iodination of penicilisorin gave 7-iodopenicilisorin which was dimerized by Pd-catalyzed homodimerization to provide (±)-bipenicilisorin. The unknown absolute configuration of naturally occurring (+)-bipenicilisorin was examined by optical resolution of the (±)-synthetic bipenicilisorin and a comparison of experimental and theoretical electronic circular dichroism (ECD) spectra. These results support the absolute configuration of the natural product to be Sa. A cytotoxic activity test of (+)-and (-)-bipenicilisorin using A549 cells revealed that (+)-1 has a lower IC50 value than (-)-1.