Knowledge of the surface composition of natural rubber (NR) latex is essential to manufacturers of latex goods. Films made from only small rubber particles (SRPs) and a mix of SRPs and large rubber particles (LRPs) differ in mechanical properties. The reason for this difference, which is still under debate, is hypothesized to be linked with biomolecules (proteins and lipids) present in the NR particle surface. In this study, we characterize the surface chemistry, particularly lipid content of the SRP and LRP, by performing investigations directly on these particles in aqueous conditions. Fluorescent probes were used to display protein and lipid affinity and analyze them in situ with steady-state fluorescence spectroscopy, fluorescence correlation spectroscopy, and fluorescence lifetime measurements. Results are atypical in showing that lipids are more abundant in LRPs than in SRPs, suggesting thicker and/or denser membranes in LRPs. The degree of membrane compacity affects rigidity, influences biomolecular interactions, and might impact natural rubber coagulation. These results provide additional insights into colloidal behavior of NR for more efficient industrial applications.