Background: HIV/AIDS is a widespread public health problem in India and rest of the World, causing severe morbidity and mortality. Occurrences of opportunistic infections (OI’s) in HIV/ AIDS correlate with their immune status. Infectious vaginitis is commonly caused by bacteria, fungi, and parasites. In general, the vaginal acidity limits the development of these infections, but the risk increases with a decrease in the immune status. Vaginitis is the most common and important OI in HIV seropositive women. The cause of concern is the recurrent and multiple episodes of vaginitis, and the emergence of drug resistance. In the general population, almost 70% of women experience at least one episode of vaginitis in their lifetime, making the condition even worse in HIV infected women resulting in recurrent and chronic infections. Knowledge of vaginitis in HIV seropositive women is inadequate. Aim: Hence this study aims to evaluate the etiology of vaginitis among HIV seropositive women before and after initiation of highly active antiretroviral therapy (HAART). Materials and Methods: A cross-sectional and prospective study was carried out between July 2013 and May 2017 at the antiretroviral therapy centre attached to the Rural Medical College (RMC), Loni, Maharashtra, India. A total of 584 HIV seropositive women, who were clinically diagnosed as suffering from vaginitis were included in the study. Among them, 18 patients lost their lives and were excluded from the study. High vaginal swabs, epithelial scrapings, and vaginal discharge were collected from all the subjects. All samples were processed for microscopy (wet mount, KOH mount, and grams staining), Amsel’s score, Nugent’s criteria, culture, and antibiotic susceptibility testing using standard laboratory methods. Statistical analysis was done using Microsoft word, and Excel. Results: Among the 566 patients followed up for a year, the incidence of vaginitis was observed predominantly in the age group of 24-35 years. 46% of the patients had bacterial vaginosis, and 22.6% suffered from vaginal candidiasis. The occurrence of vaginitis was comparatively more before initiation of antiretroviral therapy (61.1%), than after antiretroviral therapy (38.8%). Occurrence of bacterial vaginitis (58.8%) was noted to reduce after the initiation of antiretroviral therapy (42.5%). Vaginal candidiasis (63.2%) was observed to be significantly reduced after the initiation of HAART (36.7%). Microscopic examination of the vaginal discharge using Amsel’s criteria revealed the presence of vaginitis before 49 (31%) and after initiation of HAART 26 (23%) and the Nugent score showed that 84 (53%), and 61 (55%) had signs of vaginitis before and after initiation of HAART respectively. The culture results revealed 265 bacterial and 128 fungal isolates. Among the 265 bacterial isolates 156 (58.8%) were isolated before starting HAART and 109 (41.1%) were recovered after initiation of ART. Staphylococcus predominated the bacterial cause of vaginitis, and Candida albicans was the most frequent cause of vaginitis both before and after initiation of HAART. 16 (2.8%) patients showed the presence of MRSA. Conclusion: This study clearly demonstrates that there is an increased prevalence of vaginitis among the HIV infected population, both before and after initiation of HAART. Regular screening for the cause of vaginitis, initiation of appropriate antimicrobial therapy could contribute to better quality of life among HIV infected patients.