Root canal infections represent a serious challenge to the success of endodontic treatment. The most commonly used antimicrobial irrigants, such as sodium hypochlorite (NaOCl), have certain limitations, while endodontic biofilms pose a significant microbiological complexity in the endodontic field. Silver nanoparticles (AgNPs) have emerged as a promising irrigant option in root canal treatments; however, few studies are focusing on endodontic biofilms. This work aimed to evaluate the antimicrobial and anti-adherence properties of AgNPs against clinically isolated bacteria taken directly from patients with various pulp and periapical diseases. AgNPs of two sizes were synthesized and characterized. The bactericidal and anti-adherence activities of AgNPs were evaluated through microbiological assays using experimental in vitro and ex vivo tests on oral biofilms taken from patients with symptomatic apical periodontitis (AAP) and pulp necrosis (PN). NaOCl solution was used as the gold standard. The size of AgNPs was uniformly distributed (13.2 ± 0.4 and 62.6 ± 14.9 nm, respectively) with a spherical shape. Both types of nanoparticles exhibited good antimicrobial and anti-adherence activities in all microbiological assays, with a significant difference from NaOCl for in vitro and ex vivo models ( < 0.05). The inhibitory activity of AgNPs is mainly related to the type of microbiological sample and the exposure time. The antibacterial substantivity of both nanoparticle sizes was time-dependent. AgNPs may represent a promising antimicrobial option as an endodontic irrigant during conventional root canal treatments to prevent and control endodontic infections.