The aim of this study was to evaluate changes in the thickness of the peripapillary retinal nerve fiber layer (pRNFL) in children with a diagnosis of juvenile idiopathic arthritis (JIA) who were positive for human leukocyte antigen (HLA)-B27, treated for the first episode of unilateral acute anterior uveitis (AAU). This retrospective study included 41 children (aged 5 to 14 years; mean age 8.32 ± 2.4 years) with HLA-B27 positivity and unilateral JIA-AAU, and 40 healthy children. Optical coherence tomography (OCT) imaging was performed during active inflammation and subsequent noninflammatory phases (6 months after the resolution of inflammatory symptoms in the anterior segment of the eye). There was a marked difference in mean pRNFL thickness between eyes with AU in the active phase, unaffected fellow eyes and the control group (110.22 ± 5.95 μm, 102.39 ± 4.39 μm and 95.83 ± 8.84 μm, respectively; p < 0.001). The thickness of pRNFL in eyes with AU in the active phase in all sectors was greater compared to unaffected fellow eyes (p < 0.001) and normal eyes (p < 0.001). In addition, it was demonstrated that pRNFL thickness was significantly increased in the superior and temporal sectors in the unaffected fellow eyes compared to the control group (128.73 ± 13.16 μm vs. 121.48 ± 13.35 μm and 71.37 ± 4.02 μm vs. 64.98 ± 9.12 μm, respectively). Even during the inactive phase, eyes with AU, compared to the healthy control group, had significantly greater pRNFL thickness in the inferior sector (129.78 ± 11.98 μm vs. 122.3 ± 14.59 μm; p = 0.018), along with the temporal sector (70.88 ± 5.48 μm vs. 64.98 ± 9.12 μm; p = 0.001). An increase in pRNFL thickness in children with unilateral JIA-AAU who were positive for HLA-B27 antigen can be observed in both eyes compared to healthy controls, and this change may persist even after the inflammatory symptoms have resolved. Measurements of pRNFL thickness resulting from JIA-AU-associated glaucoma should be performed during quiescent periods to avoid subclinical changes in pRNFL thickness caused by inflammation. However, when reviewing the results, it should be noted that changes in pRNFL parameters may be present despite evidence of a resolution of inflammation.