Mostly studied as a zoonosis, leptospirosis is also an environment-borne infection and most human cases originate from soil or water contaminations. Yet, only few studies have been interested in the survival of pathogenicLeptospirain freshwater. In this study, water microcosms were designed to evaluate the survival and virulence ofLeptospiraspp. for 2 years. Four commercial bottled drinking waters and a non-ionized water, all previously filter-sterilized, were studied. Either one of twoLeptospira interrogansstrains, oneLeptospira borgpeterseniistrain, or a saprophyticLeptospira biflexawas inoculated in these waters under nutrient-deprived conditions. Molecular, microscopic and cultural approaches were used to studyLeptospirasurvival. Direct virulence of the pathogens was assessed using animal challenge without re-culturing. Our results confirmed the capacity of pathogenicLeptospirato survive for more than a year in water. In addition, we showed the ability ofL. interrogansin nutrient-deprived conditions to directly cause systemic infection in susceptible animals. To our knowledge, this is the first report of direct infection of a susceptible host withLeptospirafollowing a long starvation and survival period in nutrient-deprived water. Our results also suggest thatLeptospiraturned into a physiological "survival" state in harsh freshwater conditions. These data are of prime importance considering that freshwater is a major source ofLeptospirainfections. Environmental survival and virulence of pathogenicLeptospiraspp. are becoming a crucial challenge to determine the environmental risk and adopt relevant prevention and control strategies.