<p>Colonization by bacteria and fungi has been shown to reduce hatching success of sea turtle nests. Presence of microbial pathogens is commonly associated with egg failure, and in some species, it has even been shown to cause up to 90% nest mortality. Developing sea turtle embryos can only rely on non-specific defenses, such as the eggshell and the proteins present in the egg albumen. For a long time, it has been suggested that the fluid in which the eggs are coated during oviposition may contain antimicrobial properties that might protect eggs against potential pathogens that are found in the egg chamber. This study aimed to evaluate the antimicrobial properties of the cloacal fluid of loggerhead sea turtles. Cloacal fluid samples were collected at the Wassaw National Wildlife Refuge, Georgia during the 2021 nesting season (June-July). Protein fractionation of the samples was carried out using commercially available ultracentrifugation devices of 3K, 10K and 30K molecular cut-off weights. A microplate-based turbidimetric growth inhibition assay recorded the antimicrobial activity of the peptide fractions and the crude extract. We found that the cloacal fluid of loggerheads has antimicrobial properties against <i>Serratia marcescens</i> and <i>Morganella morganii</i>, and some mild action against <i>Pseudomonas aeruginosa</i>. Results obtained from the microplate-based turbidimetric assay were confirmed by assessing viability of cells upon 24 hours of exposure to the fluid. Furthermore, this study found that the cloacal fluid contains proteins with molecular weight ranging from approximately 5 to 250 kDa, and that proteins with higher molecular weights (MW>30kDa) are found in greater abundance. Further studies are needed to understand the mechanism of action of the proteins and peptides present in the cloacal fluid to potentially develop antimicrobial compounds that can be used to increase hatching success globally.</p>