Leptospires can persist for months in nutrient-poor aqueous environments prior to transmission to a mammalian host. Interactions with environmental bacteria and biofilm formation are possible mechanisms of persistence of leptospires in the environment. Bacteria isolated from rivers in the Ecuadorian rainforest were tested for their ability to support leptospiral viability. We found that co-culture with Sphingomonas spp., but not Flavobacterium spp. or Delftia spp., enabled survival of L. biflexa and L. meyeri for up to a year in distilled water. We also found that L. interrogans biofilms formed in distilled water contained viable organisms that rapidly dispersed into the planktonic phase in the presence of nutrients in serum or EMJH medium. These data inform our understanding of leptospiral survival strategies that enable long-term persistence in nutrient-poor conditions yet allow rapid mobilization when nutrients become available.