Oat (Avena sativa L.) crop constitutes a rich source of biologically active secondary metabolites. Most of these compounds act as chemical signals and defense metabolites and constitute a potential source for the development of control methods for specific diseases. Polyamines are low molecular organic cations involved in various physiological events, particularly those related to abiotic stress responses, albeit recently their potential in disease resistance has been investigated. In this work we monitored the polyamine content in leaves of both resistant and susceptible oat cultivars in response to Blumeria graminis f.sp. avenae (Bga, host interaction) and with Blumeria graminis f.sp. hordei (non-host interaction). Our results show significant differences between the resistant and susceptible cultivars for specific free polyamine levels, and also with respect to the non-host interaction at crucial stages of the infection process. In addition, polyamine degradation products, such as 1.3-diamino propane increased following pathogen challenge, suggesting a role for reactive oxygen species derived from this pathway in resistance. Exogenous application of polyamines to leaf surface increased penetration resistance of oat against Bga. Overall, data support both, a direct and indirect role for polyamines in resistance in host and non-host interactions, in responses of oat against appropriate and inappropriate powdery mildew formae speciales.