This paper reports the development and optimization of micro-matrix solid-phase dispersion (micro-MSPD) of nine polychlorinated biphenyls (PCBs) in mussel samples (Mytilus galloprovincialis) by using a two-level factorial design. Four variables (amount of sample, anhydrous sodium sulphate, Florisil and solvent volume) were considered as factors in the optimization process. The results suggested that only the interaction between the amount of anhydrous sodium sulphate and the solvent volume was statistically significant for the overall recovery of a trichlorinated compound, CB 28. Generally most of the considered species exhibited a similar behaviour, the sample and Florisil amounts had a positive effect on PCBs extractions and solvent volume and sulphate amount had a negative effect. The analytical determination and confirmation of PCBs were carried out by using GC-ECD and GC-MS/MS, respectively. The method was validated having satisfactory precision and accuracy with RSD values below 6% and recoveries between 81 and 116% for all congeners. The optimized method was applied to the extraction of real mussel samples from two Galician Rías.