Silver nanoparticles (AgNPs) are commonly used in commercial and medical applications. However, AgNPs may induce toxicity, extracellular matrix (ECM) changes and inflammatory responses. Fibroblasts are key players in remodeling processes and major producers of the ECM. The aims of this study were to explore the effect of AgNPs on cell viability, both ex vivo in murine precision cut lung slices (PCLS) and in vitro in human lung fibroblasts (HFL-1), and immunomodulatory responses in fibroblasts. PCLS and HFL-1 were exposed to AgNPs with different sizes, 10 nm and 75 nm, at concentrations 2 µg/mL and 10 μg/mL. Changes in synthesis of ECM proteins, growth factors and cytokines were analyzed in HFL-1. Ag10 and Ag75 affected cell viability, with significantly reduced metabolic activities at 10 μg/mL in both PCLS and HFL-1 after 48 h. AgNPs significantly increased procollagen I synthesis and release of IL-8, prostaglandin E2, RANTES and eotaxin, whereas reduced IL-6 release was observed in HFL-1 after 72 h. Our data indicate toxic effects of AgNP exposure on cell viability ex vivo and in vitro with altered procollagen and proinflammatory cytokine secretion in fibroblasts over time. Hence, careful characterizations of AgNPs are of importance, and future studies should include timepoints beyond 24 h.