The interplay between nanotechnology and pathogens offers a new quest to fight against human infections. Inspiring from their unique thermal, magnetic, optical, or redox potentials, numerous nanomaterials have been employed for bacterial theranostics. The past decade has seen dramatic progress in the development of various nanoantimicrobials, which demands more focus on their safety assessment. The present review critically discusses the toxicity of nanoantimicrobials and the role of key features, including composition, size, surface charge, loading capability, hydrophobicity/philicity, precise release, and functionalization, that can contribute to modulating the effects on microbes. Moreover, how differences in microbe's structure, biofilm formation, persistence cells, and intracellular pathogens bestow resistance or sensitivity toward nanoantimicrobials is broadly investigated. In extension, the most important types of nanoantimicrobial with clinical prospective and their safety assessment are summarized, and finally, based on available evidence, an insight of the principles in designing safer nanoantimicrobials for overcoming pathogens and future challenges in the field is provided.