Introduction Amoebic gill disease (AGD) is an emerging disease in North European Atlantic salmon (Salmo salar) aquaculture caused by the amoeba Paraamoeba perurans. Non-optimal environmental conditions such as increasing water temperature may affect AGD progession. To understand the role of predisposing environmental conditions on the biology of the parasiteand on the host physiological and immune response, different clonal cultures of P. perurans and AGD infected Atlantic salmon were exposed, respectively, in vitro and in vivo to two different temperatures, 10oC and 15oC. Methodology Differences in growth rate among P. perurans clonal cultures were analyised using a negative binomial analysis in R (R software, version 3.0.1). 16S MiSeq analysis was also performed to charaterise the changes in bacterial communities present in the in vitro cultures at the two different temperatures. Atlanticsalmon smolts were exposed in vitro to the same temperatures used for the in vitro experiment and to initial concentration of 500 cells/l of a clonal culture of P. perurans for 3 weeks. Analysed of cortisol, glucose and lactate concentration inplasma samples, and gene expression related have been performed at different time points after the challenge. Results Negative binomial analysis showed significant differences in growth rate among clonal cultures over time and the fraction of amoebae in suspension in seawater showed a higher increase over time at 10oC, whereas the fraction of amoebae attached to the malt yeast agar (MYA) showed a higher increase at 15oC. Atlantic salmon exposed to P. peruransdid not show clear significant increase of cortisol, glucose, and lactate levels in plasma between the two temperatures in AGD-affected fish, while histopathology showed significant differences between the two temperatures. Gene expression analysis showed significant up regulation of IL4/13 isoforms in gills at both temperatures in comparison to control fish. Conclusions The present rate from the temperature dependent AGD infection and in vitro P. perurans growth rate studies hint that increased AGD outbreaks during summer periods may not be solely due to increased thermal stress in fish but also to increased amoedae attachment at 15oC which will cause an increased gill pathology.