Background and objectives: Ear infections are a serious public health issue in developing countries. Gram-positive and gram-negative Bacteria are both capable of developing biofilms on medical equipment and earphones; however, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Enterococcus faecalis are the most prevalent types and may involve in ear infections. Since there was no such study in our region about ear set association with biofilm formation in patients with ear infections, this study was accomplished. The aim of the study: Therefore, this study was suggested in order to evaluate the relationship between the continuous use of Ear set with biofilm formation and ear infections among handlers. Patients, Materials and Methods: This study was carried out in Kirkuk City (The urban region only) from the 15th of January 2023 to the 23rd of May 2023, done on a total number of (168) participants. The total number of the patients who were Ear set users enrolled for the current study was (120), divided into two groups including (60 patients with biofilm positive (including 27 male and 33 female patients respectively) and 60 patients with biofilm negative (including 23 male and 37 female patients separately), with age, ranged from 20 to 60 years old, who were attended at Kirkuk Teaching Hospital. The control group (non-Ear set users) of the presented study was a total number of (48) including (18) males and (30) females. The serological tests included: the estimation of Human GR, SOD, and MDA antioxidants levels through using of the ELISA technique for all the participants, and the biochemical tests included measuring Vitamin D3 levels by using the iChromaTM technique, while the microbial tests were done through using of VITEK 2 compact system. Biofilm formation was detected by using the Congo red agar method. All the steps of methods are conducted based on the manufacturer's instructions. The bacterial growth among the patients group was (22.5%) of Staphylococcus aureus and (14.16%) of Pseudomonas aeruginosa, while the control group revealed (8.33 %) of Staphylococcus aureus and (4.16%) of Pseudomonas aeruginosa. The age range (20-30) years showed the highest rates among the infected patients, with male patients being more vulnerable to the infections. The mean levels of vitamin D3, human GR, SOD, and MDA decreased significantly in patients in matching to control groups. Conclusions: As a conclusion, Staphylococcus aureus and Pseudomonas aeruginosa are more prevalent agents involved in ear infections. Male patients are more susceptible patients to contracting ear infections. Vitamin D3, Human GR, SOD, and MDA antioxidants levels decline in patients with ear infections.