Abstract: Studies investigating viral ecology have mainly been conducted in temperate marine and freshwater habitats. Fewer reports are available on the often less accessible “extreme environments” such as hot springs. This study investigated prokaryotic- and virus-like particles (VLP) associated to hot springs, themselves situated in cold environments of the Southern Hemisphere (i.e. in the French Southern and Antarctic Lands). This was performed by examining their abundance in hot springs and surrounding temperate seawater using both epifluorescence microscopy (EFM) and flow cytometry (FCM), which was applied for the first time to such ecosystems. On one hand, prokaryotic abundances of 4.0 × 105–2.2 × 106 cell mL−1 and 7.0 × 104–2.8 × 106 cell mL−1 were measured using EFM and FCM, respectively. The abundances of virus-like particles (VLP), on the other hand, ranged between 9.8 × 105 and 7.5 × 106 particles mL−1 when using EFM, and between 1.3 × 105 and 6.2 × 106 particles mL−1 when FCM was applied. A positive correlation was found between VLP and prokaryotic abundances, while the virus-to-prokaryote ratio was generally low and ranged between 0.1 and 6. In parallel, samples and culture supernatants were also visualised using transmission electron microscopy. For this, enrichment cultures were prepared using environmental samples. Both raw sample and enrichment culture—supernatants were analysed for the presence of VLPs. Observations revealed the presence of Caudovirales, membrane vesicles and possibly a new type of virion morphology, associated to members of the order Thermotogales, a thermophilic and anaerobic bacterium.