AbstractDecline phenomena of shrub species such as Quercus coccifera and Retama raetam have occurred throughout Tunisian forests since 2012. These evergreen shrubs have long been regarded for their medicinal and ecological interests. Therefore, their preservation as valuable forest resources is of great interest. However, information regarding aetiology of this disease is still scarce. Hence, the aim of this study was to identify and characterize the causal agents associated with disease symptoms in two Tunisian forests. Thirty‐eight isolates were obtained from symptomatic Q. coccifera and R. raetam twigs. Morphological characterization and phylogenetic analysis of the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster and partial sequence of the translation elongation factor 1‐alpha gene (tef1‐α) allowed the identification of three Diplodia species namely Diplodia africana, D. seriata and D. pseudoseriata. Our findings revealed that the incidence of Diplodia species was significantly correlated to the altitude, the temperature and the rainfall. Pathogenicity test showed that all Diplodia isolates are pathogenic. However, D. africana revealed to be the most aggressive species toward R. raetam. These findings were the first record of D. seriata as fungal pathogen associated with Q. coccifera dieback and D. pseudoseriata and D. africana on R. raetam in Tunisia.