To understand the clinical course of human adenoviral (HAdV) conjunctivitis for establishing a better treatment regimen, thirty-eight eyes of 19 patients with HAdV-54 conjunctivitis for less than one week from onset were evaluated for clinical signs and symptoms and DNA copy numbers. It is observed that a viral load of 10⁴–10⁵ is required to develop symptoms of HAdV conjunctivitis, as symptoms were present in all the eyes whose viral load was ≥10⁴ at least once during the course. Next, it is observed that asymptomatic infections in contralateral eyes are common as the virus was detected in most eyes that did not develop conjunctivitis. Furthermore, there was no rapid decrease of the viral load in healed eyes, on the contrary, the viral load in healed eyes on day 15 was significantly higher than in unhealed eyes. This was likely due to corticosteroid instillation that rapidly alleviated symptoms but prolonged the duration of viral shedding. Recently, combination treatment with iodine and corticosteroids has been recommended for HAdV conjunctivitis. Assessing changes in viral load as well as clinical symptoms would be helpful to better understand the clinical course of this disease.