Objectives: This study aimed to investigate short-term and long-term impact of avocado consumption without caloric restriction on the gut microbiota of free-living adults with abdominal obesity. Methods: The Habitual Diet and Avocado Trial (HAT) was a 26-week, multi-center, randomized, controlled trial involving 1008 individuals with abdominal obesity. Participants were randomly assigned to the Avocado Supplemented Diet Group (AVO), receiving one avocado per day, or the Habitual Diet group (HAB), maintaining their usual dietary habits. Fecal samples were collected at baseline, week 4 and week 26 from a subset of participants recruited at a University of California Los Angeles site (n = 230). Fecal microbiota was assessed with shotgun metagenomics sequencing. Alpha diversity was assessed using the Chao1 and Shannon indices; beta diversity was assessed using Bray–Curtis dissimilarity with significance determined by repeated measures permutational multivariat analysis of variance. Potential association of intervention at week 4 and 26 with alpha diversity, species and metabolic pathways was examined using linear mixed effect models. Results: Compared to the HAB group, the AVO group had higher alpha diversity by 4 weeks, which persisted through the 26-week study period. Exploratory analysis based on healthy eating index-2015 (HEI-2015) indicated that participants with a low HEI score at baseline (≤52.7), had an increase in alpha diversity in the AVO group vs. HAB group. The AVO group had a significant change in beta diversity at week 26 compared to the HAB group. At the species level, the AVO group had significantly increased Faecalibacterium prausnitzii and Bacterium AF16_15 at week 26 compared to the HAB group. Functional analysis showed no significant difference in metabolic pathways between the HAB and AVO groups. Conclusions: Our findings document a potentially favorable effect of avocados on gut microbiota diversity. The prebiotic potential of avocados is more pronounced in individuals with a low diet quality score. This trial is registered at clinicaltrials.gov as NCT03528031 (https://clinicaltrials.gov/study/ NCT03528031).