Penicillin binding proteins (PBPs) are involved in the biosynthesis of the peptidoglycan layer constitutive of the bacterial envelope. They have been targeted for more than half a century by extensively derived molecular scaffolds of penicillins and cephalosporins. Streptococcus pneumoniae resists the antibiotic pressure by inducing highly mutated PBPs that can no longer bind the beta-lactam containing agents. To find inhibitors of PBP2x from Streptococcus pneumoniae (spPBP2x) with novel chemical scaffold so as to circumvent the resistance problems, a hierarchical virtual screening procedure was performed on the NCI database containing approximately 260000 compounds. The calculations involved ligand-based pharmacophore mapping studies and molecular docking simulations in a homology model of spPBP2x from the highly resistant strain 5204. A total of 160 hits were found, and 55 were available for experimental tests. Three compounds harboring two novel chemical scaffolds were identified as inhibitors of the resistant strain 5204-spPBP2x at the micromolar range.