Background Infectious Bovine Keratoconjunctivitis (IBK), commonly known as pinkeye, is one of the most significant diseases of beef cattle. As such, IBK costs the US beef industry at least 150 million annually. However, strategies to prevent IBK are limited, with most cases resulting in treatment with antibiotics once the disease has developed. Longitudinal studies evaluating establishment of the ocular microbiota may identify critical risk periods for IBK outbreaks or changes in the microbiota that may predispose animals to IBK. Results In an attempt to characterize the establishment and colonization patterns of the bovine ocular microbiota, we conducted a longitudinal study consisting of 227 calves and evaluated the microbiota composition over time using amplicon sequence variants (ASVs) based on 16S rRNA sequencing data and culture-based approaches. Beef calves on trial consisted of both male (intact) and females. Breeds were composed of purebred Angus and composites with varying percentages of Simmental, Angus, and Red Angus breeds. Average age at the start of the trial was 65 days ±15.02 and all calves remained nursing on their dam until weaning (day 139 of the study). The trial consisted of 139 days with four sampling time points on day 0, 21, 41, and 139. The experimental population received three different vaccination treatments (autogenous, commercial (both inactivated bacteria), and adjuvant placebo), to assess the effectiveness of different vaccines for IBK prevention. A significant change in bacterial community composition was observed across time periods sampled compared to the baseline (p < 0.001). However, no treatment effect of vaccine was detected within the ocular bacterial community. The bacterial community composition with the greatest time span between sampling time periods (98d span) was most similar to the baseline sample collected, suggesting re-establishment of the ocular microbiota to baseline levels over time after perturbation. The effect of IgA levels on the microbial community was investigated in a subset of cattle within the study. However, no significant effect of IgA was observed. Significant changes in the ocular microbiota were identified when comparing communities pre- and post-clinical signs of IBK. Additionally, dynamic changes in opportunistic pathogens Moraxella spp. were observed and confirmed using culture based methods. Conclusions Our results indicate that the bovine ocular microbiota is well represented by opportunistic pathogens such as Moraxella and Mycoplasma. Furthermore, this study characterizes the diversity of the ocular microbiota in calves and demonstrates the plasticity of the ocular microbiota to change. Additionally, we demonstrate the ocular microbiome in calves is similar between the eyes and the perturbation of one eye results in similar changes in the other eye. We also demonstrate the bovine ocular microbiota is slow to recover post perturbation and as a result provide opportunistic pathogens a chance to establish within the eye leading to IBK and other diseases. Characterizing the dynamic nature of the ocular microbiota provides novel opportunities to develop potential probiotic intervention to reduce IBK outbreaks in cattle.