Small molecule drugs have been instrumental in our fight against diseases, beginning when some of the earliest ancient cultures used plant remedies to treat illnesses. More recently, the skill of isolating and characterizing the active ingredients within bioactive plants and microbial extracts has led to the isolation and design of potent drugs such as morphine, prontosil, and penicillin. Modern medicine still relies heavily on the study of small molecules, often inspired by naturally occurring compounds. In this thesis, synthetic compounds and natural product extract libraries were screened for biological activity against HIV-1 and cancer cell line models; two relevant diseases with unmet therapeutic needs. The research discussed here is presented over three chapters. In chapter two, the synthetic isopropyl ether azaBINOL derivative B#24, was found to show low micromolar level inhibition (∼7 µM) of HIV-1 by acting on the RNase H activity of the viral reverse transcriptase enzyme. In chapter three, microbial extract library screening and bioactivity-guided fractionation of a Streptomyces sp. bacterium resulted in the first report of nanomolar anti-HIV activity from the natural compound antimycin A8a (3.1) without associated cytotoxicity. In chapter four, a target-based screening platform to discover and assess compounds with DNA-binding properties was designed and implemented utilizing biolayer interferometry. This resulted in the identification, and binding-guided isolation of bacterial metabolites including echinomycin (4.7), actinomycin V (4.8), chartreusin (4.9), and lumichrome (4.10). In summary, screening small molecule libraries, both from synthetic and natural sources, is successful if diverse chemistry, sensitive assays, and a repertoire of analytical techniques are utilized.