Staphylococcus aureus infections represent a clinical challenge due to their propensity to form biofilms and the increasing prevalence of antibiotic resistance. The ability of S. aureus to form biofilm affects clinical outcome, but techniques to study extracellular matrix (ECM) in S. aureus biofilms are lacking. Here, we present an agar-based method in which the optotracer EbbaBiolight 680 (Ebba680) is used to visualize ECM formation alongside evaluation of colony growth dynamics in agar colonies. As models for colony biofilms, we use drop inoculation for macrocolony formation or spread-plating for single-cell derived colonies. Kinetic fluorescence spectroscopy combined with time-lapse microscopy showed bright fluorescence signals, revealing different spatial-temporal appearance of ECM in macrocolonies versus single-cell derived colonies. In contrast, the microstructure was conserved between the two types of colonies. Detailed characterization of the biofilm microstructures by confocal microscopy revealed Ebba680 binding targets interspersed between cells as well as in a cap-like structure formed on the outer surface of the biofilm. Accessory gene regulator (agr) controlled expression of Ebba680 binding target(s) and the binding of Ebba680 to synthetic fibrillated phenol soluble modulins (fPSMs) suggests these functional amyloids act as targets for Ebba680 in the biofilm ECM. By upgrading ColTapp, an application developed for colony radius quantification, to also analyze fluorescence images, concurrent analysis of Ebba680-stained ECM and colony growth was achieved. This provided a new dimension to the assessment of colony biofilms. Detailed phenotypic characterization of clinical isolates is critical for treatment decision making, and enhanced screening which includes ECM as presented here has potential to facilitate treatment decisions in problematic staphylococcal infections.