Chilhuacle negro chili (Capsicum annuum L.) is an ancient Mexican landrace that is deeply linked to the culinary heritage of the country. Because of the high profitability and uniqueness of this crop, the Universidad Autónoma del Estado de Morelos is exploring its production in controlled environments. In the crop cycles of 2018 to 2019, the production of chilhuacle negro plants was seriously affected by an unidentified pathogen causing fruit rot, which reduced its quality, yield, and market value. Therefore, the main objective of this work was to study and characterize the fruit microbiota, which could help reveal the causal agent of this disease. Using DNA metabarcoding coupled with Illumina and nanopore sequencing technologies, we collected and analyzed both healthy and infected chili fruit, along with greenhouse bioaerosols. We also explored the bacterial and fungal microbiota by using microbiological techniques to isolate some of the culturable bacterial and fungal species. Our results suggest that the seedborne fungus Alternaria alternata is activated during the maturation stage of chilhuacle negro fruit, triggering a microbiome imbalance, which may in turn enable the establishment of other opportunistic pathogenic fungi during fruit decay, such as Mucor sp. To our knowledge, this is the first study of the chilhuacle negro chili microbiome, which can shed some light on our understanding of one of the main diseases that affect this valuable crop.