Background/Objectives: This study was aimed to investigate the safety profile of traditional Montenegrin Njeguški cheese by quantifying genes associated with resistance to clinically important antibiotics. Methods: Samples of Njeguški cheese were sourced from three artisan producers in Montenegro, identified as A, B, and C, with three individual batches selected per producer. Quantitative PCR (qPCR) was performed on bacterial DNA extracted directly from samples to detect genes encoding resistance to macrolide–lincosamide–streptogramin B (MLSB) [erm(A), erm(B), erm(C)], vancomycin (vanA, vanB), tetracyclines [tet(M), tet(O), tet(S), tet(K), tet(W)], β-lactams (mecA, blaZ), aminoglycosides [aac (6′)-Ie aph (2″)-Ia], and carbapenems (blaKPC, blaOXA-48, blaNDM-1, blaGES, and blaVIM). Results: Among the MLSB resistance genes, erm(B) was detected in all samples, erm(C) was present only in those from producer B, while erm(A) was found exclusively in batch 3 from producer C. Tetracycline resistance genes were widely distributed, except for tet(O), which was absent in batch 3 from producers A and B. Regarding β-lactam resistance, both blaZ and mecA were consistently detected across all samples, with statistically significant differences observed between producers. None of the samples tested positive for vancomycin resistance genes or the aminoglycoside resistance gene, regardless of producer. Among the carbapenemase genes analyzed, blaNDM-1 was the only one detected, found in most samples from producers B and C. Conclusions: This research provides the first risk assessment of artisanal and commercial Njeguški cheese regarding antimicrobial resistance genes. The findings offer valuable insights to enhance the microbiological safety of traditional Montenegrin cheeses, supporting consumer confidence in local and international markets.
