The Ōmura research group of the Kitasato Institute has isolated multiple microorganisms over a period of five decades. The resulting collection comprises a broad spectrum of microbes, including strains producing novel and diverse compounds with biological activities. A bioassay-guided fractionation of microbial culture broths has been employed to screen the microbial collection for compounds with new biological activities. And numerous novel natural products have been discovered among the microbial metabolites produced by members of the collection. However, dereplication of already known compounds and their potential analogs is a vital part of the discovery process of new microbial natural products. Recently, it has become easy to acquire the ultraviolet (UV) and mass spectrometry (MS) spectra of many single components of microbial culture broths in combination with high-performance liquid chromatography. To achieve most effective utilization of our microbial library, new compounds from microbial culture broths were investigated by employing an approach based on the physico-chemical properties using spectral analyses such as UV and MS and color reaction, collectively designated as physicochemical (PC) screening. As a result of physicochemical screening, many new compounds were identified among the secondary metabolites of fresh isolated rare actinomycetes and Streptomyces spp. preserved for a long time as producer of biological compounds. In this review, we introduce the Kitasato microbial library and the new compounds discovered from the library by PC screening.