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Simple Summary: In Italy, chickens are used for egg production and as courtyard/domestic animals and consequently veterinarians need to know their general and specialist characteristics. One key area is normal ocular measurements in order to understand any pathological changes affecting the eyes. For an accurate diagnosis and better management of ophthalmic diseases in chickens, this paper describes the normal values for the evaluation of ocular tear production, intraocular pressure, and biometric measurements of the eyes and on the microbial and cultural flora normally present in the conjunctival sac in two Italian chicken breeds. Given the abundance of chickens in Italy, it is important for veterinarians to know the normal state of chickens' eyes in order to identify any ophthalmic pathological changes. The aim of this study was to determine the normal values of select ocular parameters and to evaluate conjunctival microflora in two Italian chicken breeds. Sixty-six healthy chickens underwent a complete ophthalmic examination, which included a phenol red thread test (PRTT) for the evaluation of tear production and the assessment of intraocular pressure by rebound tonometry. B-mode ultrasound biometric measurements and conjunctival microflora identification were also performed in twenty-seven chickens. Mean PRTT was 23.77 ± 2.99 mm/15 s in the Livorno breed and 19.95 ± 2.81 mm/15 s in the Siciliana breed. Mean intraocular pressure was 14.3 ± 1.17 mmHg in the Livorno breed and 14.06 ± 1.15 mmHg in the Siciliana breed. Reference ranges for morphometric parameters were reported in the two breeds. Twenty-three chickens (85.18%) were bacteriologically positive. Chlamydia spp. antigen was detected in 14.81% of chickens. No positive cultures were obtained for fungi. Normal reference range values for selected ophthalmic parameters were obtained in clinically healthy chickens, which could facilitate accurate diagnosis and better management of ophthalmic diseases in these animals. [ABSTRACT FROM AUTHOR]