The unique reproductive strategies of botryllid ascidians, which include both asexual and sexual mechanisms as well as an extensive capacity for regeneration, contribute to their fast population growth and wide-ranging ecological effects. These colonial organisms have unique ecological adaptations and responses to environmental factors, yet comprehensive comparative studies on their environmental preferences remain scarce. We conducted an experimental study to explore the asexual reproduction and regeneration response of Botrylloides niger and Botryllus humilis colonies to varying salinity (36.5–39.5 PSU) and temperature (26 ± 1–30 ± 1°C) levels. Experimental findings highlighted species-specific preferences and stress responses: B. niger demonstrated higher tolerance to elevated salinity (39.5 PSU) with optimal growth rates at 26 ± 1–30 ± 1°C, whereas B. humilis displayed a preference for lower salinity and tendencies towards vascular budding at higher temperatures (30 ± 1°C). These observations suggest potential niche differentiation and ecological success, particularly in Mediterranean conditions, implying possible coexistence without intense competition in similar habitats. This research offers insights into the adaptive mechanisms of these ascidians, shedding light on their ecological roles and potential implications in coastal ecosystems amid changing environmental scenarios.