Two different analytical methods for the determination of polycyclic musk fragrances (PMFs) in wastewater and in activated sludge were developed. PMFs in filtered water samples were determined by gas chromatography coupled with a triple-quadrupole mass spectrometer in selected reaction monitoring (SRM) mode. Activated sludge samples were extracted using an ultrasonic bath and analysed using a GC-Ion trap. The developed methods respected a linear model (R > 0.995). Detection limits of selected compounds (Celestolide, Galaxolide, Galaxolidone, Phantolide and Tonalide) varied from 1.7 to 80 ng L for water and from 0.1 ng g to 210 ng g for activated sludge considering laboratory contamination for each PMF. Recovery studies were performed on spiked water samples and, for sludges, on procedural blanks, showing recoveries above 70% for all the considered compounds, while recovery of the internal standard was always above limit of acceptance (30%). Proposed methods were used to determine PMFs concentrations in wastewaters and activated sludges of a wastewater treatment plant (WWTP) located in Northern Italy. Concentrations in the range of μg L of Galaxolide and of its metabolite Galaxolidone were detected even in the WWTP effluent. Biotransformation of Galaxolide into Galaxolidone occurred during biological treatment with the consequent release of this compound through WWTP effluents. In activated sludges, concentrations of all PMFs except Galaxolidone were one order of magnitude higher than wastewaters, as expected according to their physicochemical properties. Present wastewater treatment technologies were confirmed to not be efficient in removing PMFs from influent wastewaters since with only ≃ 30% of Celestolide and Tonalide were removed.