The effect of ozone, diode laser irradiation, and presence of teeth crowding/spacing on pain perception in orthodontic patient was tested. Overall, 76 patients [55 women and 21 men; age 35.1(6.4) years] who met the inclusion criteria participated in the study. Immediately after fixed orthodontic appliance placement, the patients were exposed to a pain relief treatment (one single session) using either 635-nm diode laser (SmartM, Lasotronix, Warsaw, Poland) or ozone therapy (OzoneDTA, Apoza, New Taipei City, Taiwan) by placing the handpieces in the area of each teeth apex and interdental papillae, from the maxillary right first molar to the maxillary left first molar. Subjects were divided into three groups: control group (G1, n = 26), ozone (G2, n = 26, exposed to ozone therapy, generator probe type 3, working time per point 5 s, 23 points, application time 1 min and 55 s), and laser group (G3, n = 25, exposed to continuous mode diode laser, 400 mW, handpiece diameter 8 mm, spot area 0.5024 cm 2 , power density per second 1.59 W/cm 2 , dose 2 J per point, time: 5 s per point, 23 points, total energy per session 46 J, application time 1 min and 55 s). The level of teeth crowding was assessed using the Lundstrom indicator. The patients received a questionnaire for pain assessment (the Numeric Rating Scale, NRS-11, grade level 0-10) and recorded at 7 time points (1 h, 6 h, and 1, 2, 3, 4, and 5 days ) after the fixed orthodontic appliance placement. The mean pain values for the diode laser, ozone, and control group were 3.6 (1.31) (95% CI, 2.95-4.25), 5.25 (3.37) (95% CI, 3.52-6.98), and 5.75 (2.40) (95% CI, 4.69-6.81), respectively. We observed lower pain values in the diode laser group compared to the control group (p = 0.0237). The use of ozone in this study did not result in significant pain reduction in comparison to control (p = 0.8040) and laser groups (p = 0.1029). There were no differences in pain perception between patients with crowded teeth and non-crowded teeth in each group (G1, p = 0.66, G2, p = 0.86, G3, p = 0.24). The use of 635-nm diode laser led to decreased pain perception; however, ozone and presence of teeth crowding/spacing did not affect the pain perception in orthodontic patients during the first 5 days after the fixed orthodontic appliance placement.