This chapter provides a comprehensive analysis of the effects of nanoparticles on gut microbiota, emphasizing their interactions and potential repercussions of their application in medical and environmental contexts. It begins with an overview of nanoparticle characteristics, such as size, shape, and surface properties, which dictate their biological interactions and technological applications. Then it delves into the critical roles of various gut microbiota and their interactions with nanoparticles, highlighting how these particles can influence health by altering microbial communities and functions. The application of nanoparticles in drug delivery systems is discussed, highlighting their potential in enhancing treatment efficacy, while also considering their toxicity and the challenges posed by their interaction with biological systems. Emerging concerns about nanoparticle safety, their environmental impact, and their regulations form a significant part of the discussion, outlining the current understanding and gaps in research. This chapter proposes microbiota-specific protocols to evaluate the safe application of nanoparticles, stressing the need for a balanced approach that maximizes therapeutic benefits while minimizing risks. This synergy aims to guide future research and regulatory efforts, ensuring that nanoparticle technologies are developed and used responsibly with a clear understanding of their interactions with the gut microbiota and the broader ecosystem.