The aims of this research were to systematically study how extraction, desorption and gas chromatography (GC) parameters affect the volatiles composition of mushrooms in headspace solid-phase microextraction (HS-SPME) analysis. The study was carried out both with reference compounds and with Cantharellus cibarius Fr. mushroom sample. The experiments were carried out with full-factorial multivariate designs. In the desorption studies, high fiber depth in the GC injector port instead of desorption time or temperature was the most important variable for maximizing peak areas. This could be a function related to the temperature gradient inside the injector as well as a faster transfer of volatiles to the column. Out of the tested fibers, divinylbenzene/carboxen/polydimethylsiloxane (DVB/Car/PDMS) was the most suitable coating for mushroom volatiles. Additionally, extraction time of 45 vs 30 min had a high effect, while doubling the sample volume had a minimal effect. 21 volatiles were identified in C. cibarius. Hexanal and 1-octen-3-ol were the most abundant volatile compounds. Overall, aldehydes and unsaturated C6–C10 alcohols and aldehydes were the most abundant compound groups. This study demonstrated that despite the wide adaptation and history of SPME, fiber injection depth, fiber coating and extraction time are important factors that should still be carefully tested as a part of method development for mushroom-type matrices. Finally, the results of this study show that it is important to look at the extracted gas chromatogram as a whole instead of focusing on a few peaks of interest.