Biofilm characterisation has traditionally focused on bacteria, with a recent rising interest in fungal biofilms. Filamentous fungi, dimorphic fungi, and yeast biofilms frequently exhibit a layered architecture of yeast, hyphal and pseudohyphal cells. Encased in extracellular matrix, these phenotypes convey increased tolerance to fluctuating environmental and anthropogenic influences. The potential therefore exists to exploit this mode of growth for innovation in real-time, in-field biomonitoring of chemical water pollution. Yeast-based biosensors offer advantages over bacterial counterparts, as eukaryotes are more readily engineered to express mammalian molecular targets as biomarkers for health risks. Although research on yeast biofilm establishment and maturation is ongoing, information on non-model organisms is limited. This study addresses this gap by using an integrated approach for fungal biofilm characterisation through static screening and non-invasive monitoring under continuous flow. Yeast strains of Saccharomyces spp., Pichia spp., Cryptococcus spp. and Rhodotorula spp. were initially assessed in a high-throughput screen for biofilm formation under static conditions. Subsequently, the biofilm growth phases of the best-performing strains were monitored under continuous flow, using the carbon dioxide evolution measurement system (CEMS) combined with BioSpec. Metabolic activity through whole-biofilm CO2 production was tracked in parallel with biomass accumulation and biofilm-derived planktonic cell production. The environmental yeast strains C. podzolicus, R. mucilaginosa, and unexpectedly, the laboratory S. cerevisiae strains, showed pronounced biofilm formation within their respective groups. Under continuous flow conditions, both C. podzolicus and S. cerevisiae established and matured as biofilms and could be monitored using CEMS combined with BioSpec, previously only validated for monitoring bacterial biofilm establishment. The novel insight into yeast biofilm dynamics informs the potential future use of yeast biofilms in application such as continuous environmental biomonitoring using engineered yeast.