Vegetable cultivation under plastic tunnels provides high-quality crop yields but negatively affects soil fertility, especially when conventional agricultural system (e.g., use of synthetic mineral fertilizer and agrochemicals) was adopted. In this regard, the use of organic amendments has been proposed as a reliable and effective approach for soil fertility recovery. The aim of this study was to assess the effects of different organic amendments and application frequency on crop yield, soil fertility and soil microbial communities. A 1-year long mesocosm experiment was performed by conditioning a soil with different organic amendment types and frequency of application, compared with the ordinary soil management based on mineral fertilizers and fumigation. Soil fertility was assessed by determining physical and chemical soil parameters, whereas microbial community functioning and structure were assessed by high-throughput sequencing of bacterial and eukaryotic rRNA gene markers and BIOLOG EcoPlates™. Compared to the organic amendment, the use of synthetic fertilizer had a higher crop production but negatively affected pH, soil organic carbon content and soil aggregation. Diversity and richness of bacteria and eukaryotic were lower in the synthetic than in the organic amendments. The addition of organic amendments promoted the growth of Acidobacteria and Gemmatimonadetes bacteria. On the contrary, members of Actinobacteria and Proteobacteria were more abundant in the soil treated with synthetic fertilizer. This study increases our current knowledge on the effect of the synthetic and organic amendment applications on crop yield, soil fertility and soil microbial community functionality.