We examined seasonal and spatial variations of dissolved free polyamines (DFPA) in the large mesotrophic prealpine Lake Zurich (Switzerland). An ion-pairing liquid chromatography method with mass spectrometric detection was optimized for the quantification of DFPA without prior concentration or derivatization. Total DFPA concentrations varied between 0.4 nM and 11 nM in winter and spring of 2015, respectively. Polyamine concentrations were highest in the epilimnion during the phytoplankton growth phase, and reflected occurrence patterns of diatoms. Putrescine consistently dominated the DFPA pool throughout the season whereas norspermidine was detected only once in spring. Incubations with 13C-labeled putrescine revealed concentration-dependent uptake rates (3.2–5 nM h−1) and short turnover times (2.4–4.4 h). In contrast, incorporation rates into biomass measured with 14C-labeled putrescine were substantially lower (0.9 nM h−1), indicating that this compound was predominantly respired. Uptake kinetics moreover suggested that putrescine in situ uptake rates were limited by low ambient concentrations. Competition assays revealed that putrescine uptake could only be inhibited by excess concentrations of another polyamine (spermidine), but not by amino acids. Bacteria affiliated with Limnohabitans and the acI lineage of Actinobacteria were identified as the taxa with the highest fraction (25–32%) of putrescine-incorporating cells. This compound was also utilized by Alphaproteobacteria, Cyclobacteriaceae, diatoms, and cyanobacteria. Overall, our data point to fast turnover of putrescine by different microorganisms, implying the importance of this substrate as an attractive energy source.