The effects of nanoscale silver (nAg) particles on subsurface microbial communities can be influenced by the presence of biosurfactants, which have been shown to alter nanoparticle surface properties. Batch and column studies were conducted to investigate the influence of rhamnolipid biosurfactant (1–50 mg L⁻¹) on the stability and mobility of silver nanoparticles (16 ± 4 nm) in batch reactors and water-saturated columns with three solution chemistries: pH = 4 and dissolved oxygen concentration (DO) = 8.8 mg L⁻¹, pH = 7 and DO = 8.8 mg L⁻¹, pH = 7 and DO = 2.0 mg L⁻¹. In batch studies, the presence of rhamnolipid (2–50 mg L⁻¹) reduced nAg dissolution by 83.3–99.1% under all pH and DO conditions. Improved nAg stability was observed when rhamnolipid was present in batch reactors at pH = 7 ± 0.2, where the hydrodynamic diameter remained constant (∼50 nm) relative to rhamnolipid-free controls (increased to >230 nm) in 48 hours. Column experiments conducted at pH 4.0 ± 0.2 demonstrated that co-injection of nAg with rhamnolipid (2, 5 and 50 mg L⁻¹) decreased Ag⁺ breakthrough from ∼22% of total applied mass in rhamnolipid-free columns to less than 8.1% in the presence of rhamnolipid and altered the shape of the nAg retention profile from a hyper-exponential to a uniform distribution. Column experiments performed at pH 7.0 ± 0.2 and DO levels of either ∼2.0 or ∼8.8 mg L⁻¹ showed that co-injection of 5 mg L⁻¹ and 50 mg L⁻¹ rhamnolipid increased nAg mass breakthrough by 25–40% and ∼80%, respectively, enhancements in nAg stability and mobility were attributed to rhamnolipid adsorption on nAg surfaces, which effectively slowed the oxidation and thus release of Ag⁺, and adsorption of rhamnolipid on the porous medium, which competed for nAg attachment sites. These results indicate that the presence of rhamnolipid significantly influenced nAg dissolution and mobility under dynamic flow conditions. A mathematical model based on modified filtration theory (MFT) accurately reproduced nAg transport and retention behavior when aggregation and reaction processes were minimal and when rhamnolipid was present, providing a tool to predict the effects of biosurfactants on nAg transport in porous media.