To phenotypically, physiologically and molecularly characterize the causal agent of olive anthracnose in the northern Tunisia and to study its genetic variability and pathogenicity. A total of 43 isolates were obtained from symptomatic olives collected from four regions in northern Tunisia. A range of morphological and physiological characteristics was recorded; and a phylogenetic study, based on the sequence analysis of both internal transcribed spacers and TUB2 gene regions, was performed. Of the 43 isolates, 41 were identified as Colletotrichum acutatum s.s, and only two were affiliated to Colletotrichum gloeosporioides s.s. Two more representative Spanish isolates, included for comparison, were identified as Colletotrichum godetiae. Using six inter-simple-sequence-repeat markers, homogeneity between isolates from different locations and within the same species was recorded. In pathogenicity and virulence studies, C. gloeosporioides s.s was found to be less virulent, while the Spanish C. godetiae isolate was significantly more virulent than the Tunisian C. acutatum s.s. Olive anthracnose in the North of Tunisia is mainly caused by C. acutatum s.s species. This is the first study of olive anthracnose in Tunisia, which combines both phenotypic and molecular approaches. Colletotrichum acutatum s.s group was recorded for the first time in the country as the causal agent of olive anthracnose. © 2016 The Society for Applied Microbiology.