The innate immune system is the first line of defense against pathogens and immunological threats. By engaging a large cassette of pattern recognition receptors (PRRs) that recognize conserved molecular motifs on pathogens, the cells of the innate immune system can robustly respond to many classes of threats, activate specific innate and adaptive immune programs, and coordinate the nature of immune protection. While prior work has begun to show that the physical basis of receptor-ligand engagement is important for effective innate immune signaling, the quantitative impact of PRR biophysics on the innate immune response are still poorly understood and characterized. Herein, we use three diverse strategies to quantify different elements of receptor-ligand biophysics during innate immune stimulation. Firstly, we use chemically linked, multi-PRR agonists to understand how ligand organization impacts innate immune signal kinetics. Then, we develop a lattice light sheet microscopy method to label and track Toll-like receptor 2 on the surface of macrophages during activation by biophysically different stimuli to quantify receptor motion parameters associated with different responses. Finally, we use fluidic force microscopy to control presentation of single bacteria to macrophages to quantify what biophysical aspects of host cell-pathogen contact are most impactful on a single-cell exposure level. Through ordered ligand structuring and direct observations of immune cell stimulation, we show that information processing in the innate immune system is ligand- and dose-dependent and that individual cells can alter their functional state in response to the physical basis of their stimulation. These observations open the door for novel stimulant design that take advantage of physical structure and behavior to model immune interactions more faithfully or program immune responses in desired ways.