Aim: The focus of the present study was to evaluate the copper ions treatment on the viability of Mycobacterium avium subsp. paratuberculosis (MAP) and other bacterial communities in cow’s milk. Methods and Results: A copper ions treatment was evaluated in naturally contaminated cow’s milk to assay MAP load and/or viability, and relative abundance of other bacterial communities. In addition, physical–chemical analyses of the milk were also performed. All analyses were carried out before and after a copper ions treatment. After copper ions treatment, pH and copper concentration markedly increased in milk; the numbers of viable MAP significantly decreased. The relative abundance of the four target phyla decreased, with the phyla Bacteroidetes and Firmicutes surviving treatment in higher proportions (4 and 2·1% of original populations, respectively). A progressively higher percentage of dead bacterial cells after 5 and 20 min copper ions treatments was found (12 and 35%, respectively). Conclusion: With the exception of some MAP-tolerant strains, we have once again demonstrated that copper ions have a significant inactivating effect on MAP as well as certain other bacterial communities found in naturally contaminated cow’s milk. Significance and Impact of the Study: This study showed a significant inactivation of both MAP and other bacteria by copper ions in raw cow’s milk, information that could be useful as a tool for MAP control.