Background Periodontal disease poses a significant oral health challenge, involving inflammatory conditions impacting tooth-supporting structures. Treponema denticola, a "red complex" organism, plays a crucial role in periodontal pathogenesis, forming biofilms in subgingival environments and contributing to dysbiosis. Antimicrobial therapy is pivotal in managing periodontal disease, requiring a nuanced understanding of susceptibility patterns exhibited by key pathogens like T. denticola. Aims and objectives This study aims to investigate the antimicrobial susceptibility and resistance profiles of Treponema denticola, a prominent bacterium in periodontal disease, by examining its responses to various antimicrobial agents commonly used in periodontal therapy. Methodology Plaque samples were meticulously collected from individuals diagnosed with periodontal disease to ensure a diverse representation of the oral microbiome. All the samples were cultured, and red complex bacteria were isolated under anaerobic culture. Treponema denticola isolates were cultured from these samples under anaerobic conditions, and molecular techniques were employed for species identification. A comprehensive panel of antimicrobial agents was selected to assess the response of Treponema denticola. In vitro antimicrobial susceptibility testing (AST) was conducted using the antimicrobial gradient method, employing a hybrid approach combining elements of disk-diffusion and dilution methods. Results Treponema denticola had exhibitedresistance to metronidazole, a commonly used antibiotic effective against anaerobic bacteria, emphasizing limitations in its applicability. However, the bacterium displayed sensitivity to tetracycline, imipenem, cefoperazone, chloramphenicol, clindamycin, and moxifloxacin, offering diverse therapeutic options. The antimicrobial gradient strip test provided detailed minimum inhibitory concentration (MIC) values, contributing to a nuanced understanding of susceptibility and resistance patterns. Conclusion This study significantly advances our understanding of Treponema denticola's antimicrobial susceptibility and resistance profiles in the context of periodontal disease. The findings underscore the importance of tailored treatment strategies and contribute to broader efforts in antimicrobial stewardship, aligning with global initiatives to combat antibiotic resistance. This research lays the foundation for more effective and personalized approaches to periodontal care, emphasizing the intricate microbial dynamics associated with periodontal health and disease.