Enterotoxigenic Escherichia coli (ETEC) is a water-borne pathogen that causes diarrhoea. ETEC expresses virulence factors such as heat labile toxin (LT), and/or heat stable toxin (ST) that cause diarrhoea. ETEC also express colonization factors that enable attachment to the host epithelium. However, many of the virulence genes that are activated when the bacteria are exposed to stressful conditions are still unknown. In this study, the main focus was to analyse the survival of ETEC strain E2265 in an inefficient nutrient and stressful condition (freshwater) for a prolonged period in order to identify genes that are involved in mechanisms for survival. To thoroughly understand these mechanisms and particular gene activation in freshwater, transcriptomic analysis of ETEC strain E2265 was performed using Illumina paired-end RNA sequencing and differential expressed genes were analyzed using the programming language R. The results showed that genes responsible for metabolic mechanisms, cell repair and biofilm formation were activated after long period incubation in order to adapt and survive in a stressful environment (freshwater). It also indicate that the ETEC strain E2265 can survive in freshwater for long periods and remain culturable. During the more extended period the plasmid borne toxins (LT and ST) remained stable in the genome but not activated by the environmental conditions apart from a short burst of upregulated expression after 2 hours of incubation in freshwater. Also, biofilm formation of E2265 was identified, which is a thin protective layer formed by group of bacteria and genes involved in biofilm formation were activated. The results suggest that ETEC strain E2265 are able to survive for months in freshwater without losing plasmid virulence genes and that genes expression are actively modulated during different phases in early stages of freshwater incubation.