Pumpkin is a highly nutritious plant, rich in valuable nutrients that benefit human health. Due to the high perishability of this fruit, the production of pumpkin juice is a practical way to use it effectively. Recently, fermented vegetable juices have been used as a dairy alternative due to their nutritional and potential probiotic properties. This study investigated the fermentation of pumpkin juice using different strains of lactic acid bacteria (LAB), with and without heat treatment. The effects of fermentation on microbial growth, pH, acidity, extract, sugars, carotenoids, polyphenols, and antioxidant properties were analyzed. The heat-treatment process did not greatly impact the dry matter content, pH, acidity, extract, or sugar content. However, it led to a reduction in carotenoid and polyphenol levels. During fermentation, there was a consistent decrease in pH and an increase in total acidity, with no noticeable differences between bacterial strains regarding their influence on these parameters. The study revealed that there were no distinctions between LAB strains in their effects on pH, acidity, and carotenoid content in fermented pumpkin juice. Nonetheless, both L. sakei and L. plantarum proved to be effective in the fermentation process, with L. sakei demonstrating greater adaptability. The expected pH, acidity, and sugar content changes were consistently observed throughout the fermentation process. Overall, results confirm the efficacy of the used Lactobacillus strains in fermenting pumpkin juice and highlight the potential impact of heat treatment on the nutritional composition of the juice. The purpose of thermal processing of pumpkin juice, which is conducted with lactic acid fermentation, is crucial for the food industry. It extends the product’s shelf life, improves its nutritional and taste profiles, and guarantees its microbiological safety.