BackgroundIn dogs, the most frequently reported mycosis associated withAspergillusspp. are respiratory infections.Systemic aspergillosis is uncommon, with reported cases been associated with severalAspergillusspecies.Aspergillus terreusspecies complex are ubiquitous organisms, unfrequently associated with local or systemic disease in animals and humans, and treatment of osteomyelitis caused by this species is usually unfavorable.Case presentationThis case report describes the case of a 5-year-old dog, referred to the Veterinary Hospital of the Faculty of Veterinary Medicine of the University of Lisbon, Portugal, with a history of lameness of the right thoracic limb. Radiographs and CT scan revealed two different lesions on right humerus and radio, which were biopsied. The samples collected were submitted to cytological and histopathological evaluation and bacterial and mycological culture. Environmental samples, including of the surgery room and of the biopsy needle were also evaluated for the presence of fungi. Regarding biopsy samples, bacterial culture was negative, but mycological analysis originated a pure culture of a fungal species later identified asAspergillus terreusby Sanger sequencing. Results were compatible with histopathologic examination, which revealed periosteal reaction and invasion of hyphae elements. Also, mycological analysis of both environmental samples evaluated were negative. The virulence profile of the fungal isolate was phenotypically characterized using specific media, allowing to reveal its ability to produce several enzymes involved in its pathogenicity, namely lipase, hemolysin and DNAse, corresponding to a Virulence Index (V. Index.) of 0.43. The patient was submitted to itraconazole therapy for 8 weeks. After 3 weeks, the patient showed significant clinical improvement, and after 6 weeks no radiographic signs were observed.ConclusionsAntifungal therapy with itraconazole can contribute to the remission of canine infections promoted byAspergillus terreuscomplex with a relevant V. Index.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12917-023-03628-x.