The issue of antibiotic-resistant bacterial infections, coupled with the rise in viral pandemics and the slow development of new antibacterial and antiviral treatments, underscores the critical need for novel strategies to mitigate the spread of drug-resistant pathogens, enhance the efficacy of existing therapies, and accelerate the discovery and deployment of innovative antimicrobial and antiviral solutions. One promising approach to address these challenges is the dietary supplementation of non-digestible oligosaccharides (NDOs). NDOs, including human milk oligosaccharides (HMOs), play a vital role in shaping and sustaining a healthy gut microbiota. Beyond stimulating the growth and activity of beneficial gut bacteria, NDOs can also interact directly with pathogenic bacteria and viruses. Their antiviral and antibacterial properties arise from their unique interactions with pathogens and their ability to modulate the host's immune system. NDOs can function as decoy receptors, inhibit pathogen growth, bind to bacterial toxins, stimulate the host immune response, exhibit anti-biofilm properties, and enhance barrier protection. However, a notable gap exists in the comprehensive assessment of in vivo and clinical data on this topic. This review aims to provide an in-depth overview of the in vivo evidence related to the antiviral and antibacterial effects of various NDOs and HMOs, with a focus on discussing their possible mechanisms of action.