<b><i>Background:</i></b> Notwithstanding the significant advances in automated imaging techniques in the past 2 decades, subjective evaluation of the optic disc still remains an important part of glaucoma propaedeutic. In places with limited resources and a high demand for ophthalmic care, anatomical evaluation of glaucoma cases often relies solely on slit-lamp-based fundus biomicroscopic examination, which is frequently performed without mydriasis. <b><i>Objective:</i></b> The aim of this study was to compare metrics related to fundus biomicroscopy examination of the optic nerve head and peripapillary retinal nerve fiber layer (pRNFL) with and without mydriasis. <b><i>Material and Methods:</i></b> Healthy individuals, patients with early glaucoma, and glaucoma suspects were prospectively enrolled. Patients were examined before and after mydriasis by three glaucoma specialists, who estimated patients’ vertical cup-to-disc ratio (CDR) and evaluated the presence of glaucomatous signs: laminar dot sign, disc hemorrhage, disc saucering, disc notching, peripapillary atrophy, localized pRNFL defect, and loss of the ISNT pattern. Main outcome measures were the intra-observer comparison, the inter-observer agreement, and the abilities to identify glaucomatous signs before and after mydriasis. <b><i>Results:</i></b> Thirty patients (60 eyes) were enrolled (mean age, 62.3 ± 11.6 years). Considering the evaluation of the three examiners, the mean vertical CDR increased from 0.41 to 0.44 (<i>p</i> = 0.02), and the median of the coefficient of variation of the measures was reduced from 0.24 to 0.11 (<i>p</i> = 0.01) after mydriasis. Regarding the inter-observer agreement evaluation, the kappa coefficient values ranged from 0.64 to 0.72 before mydriasis and from 0.71 to 0.77 after mydriasis. Dot sign and disc notching were better identified through fundoscopic examination with mydriasis compared to the nonmydriatic examination (<i>p</i> &#x3c; 0.01). <b><i>Conclusion:</i></b> Our results suggest that fundus biomicroscopy should be performed with mydriasis whenever possible, as it yells a better intra- and inter-observer agreement and improves the detection of glaucomatous signs. Moreover, examiners seem to underestimate CDR values without mydriasis. Further investigation is warranted to validate these findings by general ophthalmologists and in different populations.