Sexually transmitted infections (STIs) are a major public health problem worldwide with significant social and economic implications. Effective control and prevention strategies necessitate a thorough understanding of the prevalence, isolation, and identification of STI pathogens. The present study aims to provide a comprehensive analysis of the isolation, identification, prevalence, and antimicrobial susceptibility pattern of STI pathogens based on culture method analysis. Endocervical /vaginal swab samples from female patients symptomatic for STI were cultured on different selective and differential media and pathogens were identified by colony morphology and biochemical tests. Antimicrobial Susceptibility Test (AST) of isolated and identified culture pathogen was performed by using Kirby-Bauer disc diffusion method. Among 209 endocervical/vaginal swab samples from symptomatic patients, 126 (60.28%) tested positive and 83 (39.71%) negative. Ureaplasma spp. (n = 100) was the most prevalent isolate, constituting 79.36% of culture-positive samples, followed by N. gonorrhoea (n = 99) at 78.57%, and Mycoplasma spp. (n = 41) at 32.54% individually and in combination. AST analysis revealed erythromycin (74%), ofloxacin (69%), and roxithromycin (64%) as the most resistant antibiotics for Ureaplasma spp. N. gonorrhoea showed the highest resistance to cefixime (78.79%), followed by ofloxacin (75.76%) and erythromycin (69.7%). Azithromycin and erythromycin exhibited 100% resistance against Mycoplasma spp. The study provides information on the prevalent bacterial pathogens involved in STIs among women in Anuppur and Shahdol districts, Madhya Pradesh. Understanding the diversity, distribution patterns and antibiotic sensitivity of these pathogens is crucial for developing targeted interventions and effective prevention strategies in such resource-limited areas.