Fresh carrot juice presents nutritional and organoleptic qualities which have to be preserved. However, it is a fast perishable beverage, and its low-acidic pH promotes the development of foodborne pathogens and spoilage microorganisms. This study aims to assess the modification and variability of physicochemical and microbial indicators during storage of carrot juice, and to isolate and select microorganisms to be used as promoters of spoilage to quantify the effect of preservation treatments. To achieve that, 10 batches of carrot Daucus carota cv. Maestro juice were prepared independently, stored up to 14 days at 4 °C and analyzed. Volatile compound composition differed mainly according to the analyzed batch. During storage, an increase of the content of ethanol, ethyl acetate or 2-methoxyphenol, which are produced by different microorganisms, was noticed. Isolation of bacteria revealed Pseudomonas, lactic acid bacteria, and enterobacteria, some of them provoking odor modification of carrot juice at 4 °C. Assays in carrot juice with isolated yeasts and molds showed the ability of Meyerozyma guillermondii to induce texture modification and some isolates, e.g., Pichia guillermondii, resulted in gas production. Selected isolates able to induce spoilage are useful to test preservative treatments of fresh carrot juice under controlled conditions.