Antimicrobial drug resistance has made the treatment of microbial infections quite challenging. A Gram-positive, anaerobic, spore-forming, and toxin-producing bacillus, Clostridioides difficile infection causes diarrhea-related deaths globally. The available drugs like vancomycin and metronidazole are becoming less effective against this infection. We have designed this study to identify genes responsible for antimicrobial resistance and have a better understanding of the mutations and their impact on the antimicrobial resistance activity. The Whole Genome Sequencing data of 11 C. difficile clinical isolates was analyzed to determine novel genes playing a significant role in antimicrobial resistance mechanisms. Comparative structure analysis of wild and mutant structures of proteins and their functions provided insight into the impact of the identified mutations on antimicrobial resistance. We identified 8 genes common in all the isolates that play a vital role in drug resistance through antibiotic efflux, ribosomal protection, and antibiotic inactivation. Variations in the functional domains of tetA(P), tetM, and ermB genes were found to be the most promising novel drug targets. Our findings suggest that these novel gene mutations would be beneficial in designing new drugs to combat C. difficile infection.