Silver nanoparticles (AgNPs) have attracted increased interest and are currently used in various industries including medicine, cosmetics, textiles, electronics, and pharmaceuticals, owing to their unique physical and chemical properties, particularly as antimicrobial and anticancer agents. Recently, several studies have reported both beneficial and toxic effects of AgNPs on various prokaryotic and eukaryotic systems. To develop nanoparticles for mediated therapy, several laboratories have used a variety of cell lines under in vitro conditions to evaluate the properties, mode of action, differential responses, and mechanisms of action of AgNPs. In vitro models are simple, cost-effective, rapid, and can be used to easily assess efficacy and performance. The cytotoxicity, genotoxicity, and biocompatibility of AgNPs depend on many factors such as size, shape, surface charge, surface coating, solubility, concentration, surface functionalization, distribution of particles, mode of entry, mode of action, growth media, exposure time, and cell type. Cellular responses to AgNPs are different in each cell type and depend on the physical and chemical nature of AgNPs. This review evaluates significant contributions to the literature on biological applications of AgNPs. It begins with an introduction to AgNPs, with particular attention to their overall impact on cellular effects. The main objective of this review is to elucidate the reasons for different cell types exhibiting differential responses to nanoparticles even when they possess similar size, shape, and other parameters. Firstly, we discuss the cellular effects of AgNPs on a variety of cell lines; Secondly, we discuss the mechanisms of action of AgNPs in various cellular systems, and try to elucidate how AgNPs interact with different mammalian cell lines and produce significant effects; Finally, we discuss the cellular activation of various signaling molecules in response to AgNPs, and conclude with future perspectives on research into AgNPs.