Purpose: This study was designed to assess retinal and choroidal microvasculature in acne patients using oral isotretinoin with optical coherence tomography angiography. Materials and methods: One hundred and one eyes of 101 patients diagnosed with acne using isotretinoin and 105 eyes of 105 healthy subjects underwent retinal and choroidal assessment with the Triton swept-source deep range imaging optical coherence tomography device (Topcon Corp., Tokyo, Japan). The patients’ superficial (VDs), deep (VDd), and choriocapillar (VDc) vascular densities were examined. Foveal avascular zones in the superficial (FAZs) and deep (FAZd) layers, as well as the central macular thickness (CMT) and subfoveal choroidal thickness (SFCT), were measured. Results: In total, 32 males and 69 females were assessed. The mean duration of isotretinoin use was 81 days (range: 30–210 days). The cumulative dose of medication used was 2334 mg (range: 600–7200 mg). VDs and VDd showed no differences between the acne patients and the controls (p > 0.05). All sectors of the VDc values were significantly higher in the patients compared to the controls (p < 0.05). FAZs and FAZd were significantly higher in the patients compared to the controls (p = 0.048 and p = 0.041, respectively). The mean CMT significantly decreased in the patient group compared to the control group (p = 0.001), while the mean SFCT significantly increased (p = 0.002). The VDc superior values of the patients who used isotretinoin for more than 3 months were significantly higher than those of the patients who used the drug for less than 3 months (p = 0.01). We did not find any correlation with the total dose of isotretinoin, except for the nasal sector value of VDc (r = −0.199, p = 0.046). Conclusions: Alterations of macular microvasculature were observed in acne patients using isotretinoin. In particular, a significant increment in choriocapillaris blood flow was detected.