The large lakes south of the Alps constitute the most important lake district in Italy. In these regions, Tychonema bourrellyi was identified only recently, with anatoxin-a (ATX-a) producing populations developing with high biomasses in spring and early summer. The increasing development of T. bourrellyi and the associated risk for ATX-a exposure may have a negative impact on the use of water resources. In this regard, recent molecular tools proved to be suitable for the analysis and surveillance of toxigenic microorganisms, providing significant advances in water quality management. In this study, we designed species-specific primers based on the rbcLX genes, for the identification and cell quantification of T. bourrellyi in environmental samples, for PCR and qPCR (SYBR Green) applications. The application of this assay for environmental analyses was validated on Lake Garda samples, showing a significant correlation with densities estimated by light microscopy (LM). Moreover, densities estimated by both LM and qPCR were highly correlated with concentrations of ATX-a. This sensitive method can be used for the long-term study of T. bourrellyi population dynamics, and its fast monitoring in the alpine region, improving risk assessment evaluations and the management of waters.