Background: Anaerobic Gram-negative bacteria (AGNB) play a significant role as both pathogens and essential members of the human microbiota. Despite their clinical importance, there remains limited understanding regarding their antimicrobial resistance (AMR) patterns. This knowledge gap poses challenges in effectively managing AGNB-associated infections, as empirical treatment approaches may not adequately address the evolving resistance landscape. To bridge this research gap, we conducted a comprehensive study aimed at exploring the role of human AGNB as a reservoir of AMR. This can provide valuable insights for the prevention and management of anaerobic infections. Methods: We studied the prevalence of AMR and AMR determinants conferring resistance to metronidazole (nimE), imipenem (cfiA), piperacillin–tazobactam (cepA), cefoxitin (cfxA), clindamycin (ermF), chloramphenicol (cat) and mobile genetic elements (MGEs) such ascfiAISandIS1186 associated with thecfiAandnimgene expression. These parameters were studied inBacteroidesspp.,Fusobacteriumspp.,Prevotellaspp.,Veillonellaspp.,Sutterellaspp., and other clinical AGNB. Results: Resistance to metronidazole, clindamycin, imipenem, piperacillin–tazobactam, cefoxitin and chloramphenicol was 29%, 33.5%, 0.5%, 27.5%, 26.5% and 0%, respectively. The presence of resistance genes, viz.,nim,ermF,cfiA,cepA,cfxA,was detected in 24%, 33.5%, 10%, 9.5%, 21.5% isolates, respectively. None of the tested isolates showed the presence of acatgene and MGEs, viz.,cfiAISand IS1186. The highest resistance to all antimicrobial agents was exhibited byBacteroidesspp. The association between resistant phenotypes and genotypes was complete in clindamycin, as all clindamycin-resistant isolates showed the presence ofermFgene, and none of the susceptible strains harbored this gene; similarly, all isolates were chloramphenicol-susceptible and also lacked thecatgene, whereas the association was low among imipenem and piperacillin–tazobactam. Metronidazole and imipenem resistance was seen to be dependent on insertion sequences for the expression of AMR genes. A constrained co-existence ofcepAandcfiAgene inB. fragilisspecies was seen. Based on the absence and presence of thecfiAgene, we dividedB. fragilisinto two categories, Division I (72.6%) and Division II (27.3%), respectively. Conclusion: AGNB acts as a reservoir of specific AMR genes, which may pose a threat to other anaerobes due to functional compatibility and acquisition of these genes. Thus, AST-complying standard guidelines must be performed periodically to monitor the local and institutional susceptibility trends, and rational therapeutic strategies must be adopted to direct empirical management.