Ammonia-oxidizing Archaea (AOA) are ubiquitous throughout the oceanic water column; however, our knowledge on their physiological and ecological diversity in different oceanic regions is rather limited. Here, we report the cultivation and characterization of two novel Nitrosopumilus strains, originating from coastal surface waters of the Northern Adriatic Sea. The combined physiological and genomic information revealed that each strain exhibits different metabolic and functional traits, potentially reflecting contrasting life modes. Strain NF5 contains many chemotaxis-related genes and is able to express archaella, suggesting that it can sense and actively seek favorable microenvironments such as nutrient-rich particles. In contrast, strain D3C is non-motile and shows higher versatility in substrate utilization, being able to use urea as an alternative substrate in addition to ammonia. Furthermore, it encodes a divergent, second copy of the AmoB subunit of the key enzyme ammonia monooxygenase, which might have an additional catalytic function and suggests further metabolic versatility. However, the role of this gene requires further investigation. Our results provide evidence for functional diversity and metabolic versatility among phylogenetically closely related thaumarchaeal strains, and point toward adaptations to free-living versus particle-associated life styles and possible niche differentiation among AOA in marine ecosystems.