Background For over 50 years, affirmative action helped advance equity, diversity and inclusion (EDI) in educational institutions in the United States (U.S.). However, the recent U.S. Supreme Court decision to end affirmative action in college admissions threatens the progress toward EDI. Objective This study aimed to assess the progress in promoting gender and racial diversity within the discipline of microbiology over a 55-year period. We sought to analyze the representation of women and minority groups in faculty ranks, tenure positions, and leadership to identify disparities and trends and determine who will likely be impacted most with the end of affirmative action. Materials and methods This longitudinal retrospective study utilized publicly available and non-identifiable Association of American Medical Colleges (AAMC) data on full-time microbiology faculty from 1967 to 2021. Faculty members were categorized based on academic ranks and tenure status, while gender and racial data were also considered. Results The analysis revealed a consistent dominance of white faculty, with over 60% representation across all academic ranks throughout the study period. The Asian and female faculty representation decreased in senior academic ranks. We observed a positive trend in the annual increase of women in faculty positions, academic ranks, chairs, and tenure positions. Furthermore, Asian faculty demonstrated the most robust surge in representation. However, disparities persisted for black, Hispanic, and Native American faculty members, reflecting broader challenges in their representation. Discussion Although efforts to enhance diversity within microbiology have yielded positive results, underrepresented minority groups still face obstacles in attaining leadership positions and senior academic ranks. The diminishing proportion of women at higher academic ranks raises concerns about potential attrition or lack of promotion opportunities. The end of affirmative action poses a risk of perpetuating this trend, leading to a decline in diversity among microbiology faculty.