Technical Abstract: Secondary fermentations during the bulk storage of fermented cucumbers can result in spoilage that causes a total loss of the fermented product, at an estimated cost of $6,000 to $15,000 per affected tank. Previous research has suggested that such fermentations are the result of microbiological utilization of lactic acid and the formation of acetic, butyric, and propionic acids. The objectives of this study were to characterize the chemical and environmental conditions associated with secondary cucumber fermentations and to isolate and characterize potential causative microorganisms. Both commercial spoilage samples and laboratory-reproduced secondary fermentations were evaluated. Potential causative agents were isolated based on morphological characteristics. Two yeasts, Pichia manshurica and Issatchenkia occidentalis, were identified and detected most commonly concomitantly with lactic acid utilization. In the presence of oxygen, yeast metabolic activities lead to lactic acid egradation, a small decline in the redox potential (Eh, Ag/AgCl, 3MKCl) of the fermentation brines, and an increase in pH to levels at which bacteria other than the lactic acid bacteria responsible for the primary fermentation can grow and produce acetic, butyric, and propionic acids. Inhibition of these yeasts by allyl isothiocyanate (AITC) resulted in stabilization of the fermented medium, while the absence of the preservative resulted in the disappearance of lactic and acetic acids in a model system. Additionally, three Gram-positive bacteria, Lactobacillus buchneri, a Clostridium sp., and Pediococcus ethanolidurans, were identified as potentially relevant to different stages of the secondary fermentation. The unique opportunity to study commercial spoilage samples generated a better understanding of the microbiota and environmental conditions associated with secondary cucumber fermentations.