Harmful algal blooms (HABs) represents a major environmental concern in coastal marine ecosystems due to their impacts on human health, fisheries, aquaculture, tourism, and ecosystem functioning. In the Mediterranean Sea, blooms of toxin-producing microalgae, including species belonging to Ostreopsis sp., Alexandrium sp., and Dinophysis sp., highlighted the need for effective monitoring strategies that can support risk assessment programs and provide early warning system. Traditional light microscopy monitoring is still crucial for phytoplankton identification; however, limitations in species discrimination, particularly for cryptic and low abundant taxa remain to be faced. Molecular techniques represent powerful tools, enabling rapid, sensitive, and accurate detection of harmful microalgal species. Approaches such as quantitative PCR (qPCR), metabarcoding, and environmental DNA (eDNA) sequencing have significantly improved the capacity to characterize phytoplankton assemblages and identify potentially toxic species even in complex environmental matrices. In order to make molecular approaches effective for supporting timely responses to harmful algal bloom events, the harmonization of protocols and data sharing will be fundamental. Applications of these approaches in Mediterranean coastal environments have enhanced our understanding of HAB dynamics, also enabling the detection of resting stages of potentially toxic taxa in sediments and the identification of harmful taxa associated with emerging substrates, such as marine plastic debris, contributing to a more comprehensive assessment of species dispersal and persistence. Moreover, combining high-throughput sequencing technologies with environmental factors, predictive capabilities can be improved to support the development of early warning systems. In the current context of climate changes and anthropogenic pressures altering Mediterranean marine ecosystems, molecular monitoring represents a key component for understanding HAB occurrence and dynamics, assessing associated risks, and implementing effective mitigation strategies.