To improve the quality and organoleptic properties of fermented pine needle, the physiological parameters and microbial communities must be explored. In this study, high-throughput sequencing was used to explore bacterial and fungal communities during the fermentation process of pine needle after adding starter culture of 0.8% activated dry yeast and Lactobacillus fermentum CECT5716 and Bifidobacterium Breve M-16 V. During the fermentation, the total flavonoid (from 0.049 to 111.404 mg/L) and polyphenol (from 19.412 to 183.399 mg/L) contents increased rapidly from 0 to 15 days. The total sugar increased from 0 to 3 days fermentation with a range from 3.359 mg/mL to 45.502 mg/mL in yeast fermentation stage, and reached the maximum value on day 3. The content of total acid (39.167 g/L) and amino acid nitrogen (1.185 g/L) gradually increased during the entire fermentation process and reached a maximum on day 7 of bacterial fermentation. Firmicutes and Proteobacteria phyla were the predominant bacteria in all time periods. At the genus level, well-known Lactobacillus was the most abundant bacterial strain on day 3, followed by Gluconobacter. The Acetobacter constituted more than 50% of the total bacterial abundance on day 1, but it decreased with the fermentation process. Exploration of fermented pine needle microbial community structure will expand the knowledge about their microbiota to manipulate the microbial community and improve their quality and organoleptic properties using different microbial recipes.