Cultural-based methods of bacteria and fungi and molecular identification of bacteria were combined with parallel continuous measurements of aerosol chemical composition, number size distribution, ice-nucleating concentration, and fluorescent particle size distribution and characteristics. Measurements took place at the Helmos Hellenic Atmospheric Aerosol and Climate Change Station (HAC)2, Greece, during the 2021 CALISHTO campaign. The objective was to characterize the microorganism levels at the (HAC)2 station and further investigate the associations between bioaerosols and aerosols in atmospheric processes that play a key role in the formation of ice crystals. Very low concentrations of viable, cultivable heterotrophic bacteria (4 ± 4 CFU/m3) were measured, whereas fast-growing fungi were not affected (182 ± 86 CFU/m3) by the environmental conditions at the station. The size distribution of heterotrophic bacteria was bimodal with peaks at fine (1.1-2.1 μm) and coarse size fractions (d > 7 μm), whereas airborne fungi exhibited a monomodal distribution (2.1-3.3 μm). Bacterial populations identified using 16S rRNA correlated well (r = 0.82) with the averaged concentrations of fluorescent particles (A and C channels). Strong correlations were obtained between total bacterial and particle volume concentrations of coarser fractions (>1 μm, 0.61-0.86), suggesting their strong presence in these sizes. No correlation was found with ice nuclei (INP) (r = −0.04) and low to medium negative correlations with the organics and ions (SO42-, NH4+, NO3-, Cl-) possibly due to their relatively lower sizes. In accordance with the culture-dependent analysis, relatively low total bacterial concentrations were determined by real-time PCR, with concentrations ranging from 33.4 to 117.2 Escherichia coli GE/m3. High bacterial diversity was found with 123 bacterial Operational Taxonomy Units (OTUs) classified in 10 phyla, 16 classes, 56 families, and 78 genera. Origin of the air masses was a significant driver to bacterial communities. Enrichment of specific species such as Geodermatophilus africanus and Actinomycetales bacterium was observed during Saharan dust episodes, while in the presence of continental air masses, characteristic species such as Rhizobium sp., Corynebacterium sp., and Staphylococcus caprae had higher relative abundance. Our study provides a comprehensive analysis and quantification of the varying drivers and variability in microorganisms in high-altitude site.