Chalazion is a common infection of the meibomian gland in all ages’ eyelids. The current study sought to isolate and identify bacteria linked with chalazion in patients hospitalized to Mosul hospitals between 10/1/2021 and 1/3/2022. Samples were obtained using swabs soaked in the transport media (amies), and bacteria were identified using traditional methods with API20E. Vitek-2 and molecular approaches based on the 16 S rRNA gene verified the species-level diagnosis. Using BLAST software, the nitrogen base sequences were determined and compared to those of the NCBI. The results showed that Staphylococcus aureus was isolated by 52.2% and 62.5% among women, notably in the age (21-40) years, and by 27.8% for females and 19.2% for the same age group in males, and upper eyelid infection predominated. The infection rate in people with oily skin was 50.9% in females and 34.6% in men, with the presence of the agr A, agrC, and agrD genes in isolates that exhibited a positive result in the Congo red medium and microtiter plate.