Streptococcus pneumoniae (S. pneumoniae) remains a leading cause of respiratory and invasive diseases despite the availability of vaccines. These vaccines target a polysaccharide capsule the bacterium uses to evade the immune system. Variation of the capsule composition subdivides the organism into serotypes and influences its protective potency. It is commonly assumed that S. pneumoniae strains of the same serotype produce capsules of consistent size, despite the organism’s heterogeneity. This study challenged this assumption by analyzing clinical isolates of the same serotype. Two methods were modified to analyze the S. pneumoniae capsule: immunoblot quantification of capsular polysaccharide in bacterial lysates and light microscopy to assess capsule thickness. The dataset includes raw files for microscopy images for the dextran exclusion assay, including processed images with Regions of Interest and associated measurements. In addition, it contains raw immunoblot images, including culture metrics and densitometry results.