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Simple Summary: The ocular surface microbiome is altered in certain disease states. This study aimed to characterize the ocular surface microbiome of cattle with and without ocular squamous cell carcinoma (OSCC), a common eye disease in cattle. From a sample population of ten normal adult female cattle (twenty normal eyes) and nine adult female cattle with unilateral or bilateral OSCC lesions (ten diseased eyes, eight normal eyes), significant quantitative (real-time polymerase chain reaction (RT-PCR)) and relative abundance (16S ribosomal ribonucleic acid (rRNA) gene sequencing) bacterial microbiome alterations were identified based on disease status (normal or OSCC) and geographic location. Quantitative bacterial ocular microbiome discriminant analysis (DA) was capable of accurately categorizing samples based on disease status and geographic location. Quadratic DA, a method based on relatively inexpensive and readily accessible equipment, may be valuable for differentiating between normal and OSCC-affected cattle. The ocular surface microbiome is altered in certain disease states. The aim of this study was to characterize the bovine bacterial ocular surface microbiome (BBOSM) in the context of ocular squamous cell carcinoma (OSCC). The conjunctiva of normal (n = 28) and OSCC (n = 10) eyes of cows aged 2 to 13 years from two farms in Louisiana and Wyoming were sampled using individual sterile swabs. DNA extraction followed by 16S ribosomal ribonucleic acid (rRNA) gene sequencing and real-time polymerase chain reaction (RT-PCR) were performed to, respectively, assess the relative and absolute BBOSM. Discriminant analysis (DA) was performed using RT-PCR data, and relative abundance analysis was performed using 16S rRNA gene sequencing data. The 11 most abundant phyla in both normal and OSCC-affected cows were identified using 16S rRNA gene sequencing analysis. The relative abundance of Euryarchaeota was found to be significantly lower (p = 0.0372) in OSCC eyes compared to normal eyes. Relative abundance differences within and between geographic locations were also identified. Quadratic DA categorized samples as OSCC or normal with 100% sensitivity and 83.3–100% specificity. Relative abundance analysis identified relative BBOSM phylum alterations in OSCC. Quadratic DA can be used to accurately categorize BBOSM from normal and OSCC ocular surface samples. [ABSTRACT FROM AUTHOR]