The effect of visitor presence on zoo animals has been explored in numerous studies over the past two decades. However, the opportunities for observations without visitors have been very limited at most institutions. In 2020, the Oregon Zoo was closed, in response to the global SARS-CoV-2 (COVID-19) pandemic, from 15 March 2020 to 12 July 2020, resulting in approximately four consecutive months without visitor presence. This study aimed to quantify potential behavioral and hormonal changes expressed during two transition periods in zoo visitor attendance: the initial time period before and after closure in March 2020 and time before and after reopening in July 2020. Fecal glucocorticoid metabolite (fGM) concentrations of resident giraffes (n = 2) and cheetahs (n = 2) were tracked using enzyme immunoassay (EIA) analyses. Average fGM concentrations during the two transition periods were compared using a two-way mixed ANOVA. Additionally, twice-weekly scan sampling was used to quantify behavioral observations across the transitions, which were analyzed as individual behavior proportions. Individual behavior proportions were compared across the Zoo’s opening status and time of day using Kruskal–Wallis (H) tests. The results of our analyses showed the following outcomes: (1) significant increases in fGM concentrations for cheetahs and giraffes between the transition periods but not within them; (2) a significant increase in time spent ‘not visible’ in the cheetahs in the second transition period; and (3) increased vigilance behaviors in the giraffes immediately after the Zoo’s closure. However, the changes observed in fGM concentrations may be more strongly correlated with concomitant social changes (giraffes) and some medical events (cheetahs) rather than with the Zoo’s opening status. Nevertheless, this study was able to quantify differences in behavioral frequencies and fGM concentration in cheetahs and giraffes at the Oregon Zoo during the times of transition between visitor’s presence and absence. The results indicate that, while there was a possible, but relatively minor impact of the presence and absence of visitors on some behaviors, the differences observed in fGM concentration may have been more affected by some of the concomitant social changes and medical events that happened during the same period than by the presence or absence of visitors.