Ribosome-inactivating proteins (RIPs), enzymes that are widely distributed in the plant kingdom, inhibit protein synthesis by depurinating rRNA and many other polynucleotidic substrates. Although RIPs show antiviral, antifungal, and insecticidal activities, their biological and physiological roles are not completely understood. Additionally, it has been described that RIP expression is augmented under stressful conditions. In this study, we evaluated protein synthesis inhibition activity in partially purified basic proteins (hereafter referred to as RIP activity) from tissue extracts of Fragaria × ananassa (strawberry) cultivars with low (Dora) and high (Record) tolerance to root pathogens and fructification stress. Association between the presence of RIP activity and the crop management (organic or integrated soil), growth stage (quiescence, flowering, and fructification), and exogenous stress (drought) were investigated. RIP activity was found in every tissue tested (roots, rhizomes, leaves, buds, flowers, and fruits) and under each tested condition. However, significant differences in RIP distribution were observed depending on the soil and growth stage, and an increase in RIP activity was found in the leaves of drought-stressed plants. These results suggest that RIP expression and activity could represent a response mechanism against biotic and abiotic stresses and could be a useful tool in selecting stress-resistant strawberry genotypes.